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PubMed2004

Preimplantation genetic diagnosis for an insertional translocation carrier.

BACKGROUND: While preimplantation genetic diagnosis (PGD) is well established for carriers of reciprocal terminal translocations, reports on PGD for insertional translocation carriers are lacking. Here, we report on the PGD of an insertional translocation carrier with karyotype 46,XX,ins(14;2)(q21;q31q35). Due to the possibility of crossovers within the inserted region, rather than a single probe, four probes are required for proper embryo selection. METHODS: Probes were generated for PGD using fluorescence in situ hybridization and two PGD cycles. RESULTS: Analysis of 10 embryos revealed four embryos to be normal diploid. Two embryos were consistent with 3:1 segregation of the theoretical quadrivalent and one was consistent with 2:2 or 1:1 segregation. Furthermore, one embryo was mosaic abnormal and one remained without diagnosis. CONCLUSIONS: With increased acceptance of PGD, it is likely that more carriers of complex translocations will enter PGD programmes. The present results suggest that a careful genetic work-up of complex translocations is essential for proper embryo selection. While theoretical modelling may predict that quadrivalents will form during the meiosis of insertional translocations, experimental proof for the occurrence of quadrivalents is still lacking and more research on the meiotic process of both female and male insertional translocation carriers is warranted.

Blastocyst↗
PubMed1989

Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis.

The complete sequence of an insertion element IS900 in Mycobacterium paratuberculosis is reported. This is the first characterised example of a mycobacterial insertion element. IS900 consists of 1451bp of which 66% is G + C. It lacks terminal inverted and direct repeats, characteristic of Escherichia coli insertion elements but shows a degree of target sequence specificity. A single open reading frame (ORF 1197) coding for 399 amino acids is predicted. This amino acid sequence, and to a lesser extent the nucleotide sequence, show significant homologies to IS110, an insertion element of Streptomyces coelicolor A3(2). It is proposed that IS900, IS110, and similar insertion elements recently identified in disease isolates of Mycobacterium avium are members of a phylogenetically related family. IS900 will provide highly specific markers for the precise identification of Mycobacterium paratuberculosis, useful in defining its relationship to animal and human diseases.

Amino Acid Sequence↗
PubMed1990

A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction.

We have developed a strategy to rapidly construct DNA hybridization probes for the isolation of genes disrupted by transposon Tn5 insertions. A single oligonucleotide complementary to and extending outward from the ends of the inverted repeat of Tn5 was used to prime DNA synthesis in the polymerase chain reaction. The amplified product consisted of DNA sequences adjacent to both ends of the transposon insertion. The general feasibility of the approach was tested by amplifying pBR322 sequences from a derivative of pBR322 containing a Tn5 insertion. To amplify genomic DNA sequences flanking a Tn5 insertion in the chromosome of a Pseudomonas syringae strain, circular substrates were generated by ligating EcoRI-digested genomic DNA. Tn5 was contained intact within one such circular molecule, as the transposon does not contain sites for cleavage by EcoRI. The amplified product (approximately 2.5 kb) was used as a DNA hybridization probe to isolate the homologous fragment from a cosmid library of wild-type Pseudomonas syringae genomic DNA. This approach may be applied to the efficient isolation of sequences flanking any Tn5 insertion.

Base Sequence↗
PubMed1998

Transfer of P1 inserts into a yeast-bacteria shuttle vector by co-transformation mediated homologous recombination.

Manipulation of genomic inserts cloned into the bacteriophage P1 vector is hindered by the large size of the inserts. We have used co-transformation mediated recombination between the yeast-bacteria shuttle vector, pClasper, and various P1 clones to transfer the entire insert from the P1 into pClasper. This results in the insert being stably maintained in yeast, facilitating mutagenesis by homologous recombination. The recombinant plasmid can subsequently be transferred to and stably maintained in bacteria for efficient plasmid preparation. This method can also be applied to inserts from P1 artificial chromosome or bacterial artificial chromosome vectors.

Bacteriophage P1↗
PubMed1998

Trypanosoma brucei U insertion and U deletion activities co-purify with an enzymatic editing complex but are differentially optimized.

RNA editing, the processing that generates functional mRNAs in trypanosome mitochondria, involves cycles of protein catalyzed reactions that specifically insert or delete U residues. We recently reported purification from Trypanosoma brucei mitochondria of a complex showing seven major polypeptides which exhibits the enzymatic activities inferred in editing and that a pool of fractions of the complex catalyzed U deletion, the minor form of RNA editing in vivo . We now show that U insertion activity, the major form of RNA editing in vivo , chromatographically co-purifies with both U deletion activity and the protein complex. Furthermore, these editing activities co-sediment at approximately 20 S. U insertion does not require a larger, less characterized complex, as has been suggested and could have implied that the editing machinery would not function in a processive manner. We also show that U insertion is optimized at rather different and more exacting reaction conditions than U deletion. By markedly reducing ATP and carrier RNA and increasing UTP and carrier protein relative to standard editing conditions, U insertion activity of the purified fraction is enhanced approximately 100-fold.

Animals↗
PubMed1999

Insertion of dGMP and dAMP during in vitro DNA synthesis opposite an oxidized form of 7,8-dihydro-8-oxoguanine.

Oxidative damage to DNA bases commonly resultsin the formation of oxidized purines, particularly 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), the former being a well-known mutagenic lesion. Since 8-oxoG is readily subject to further oxidation compared with normal bases, the insertion of a base during DNA synthesis opposite an oxidized form of 8-oxoG was investigated in vitro. A synthetic template containing a single 8-oxoG lesion was first treated with different one-electron oxidants or under singlet oxygen conditions and then subjected to primer extension catalyzed by Klenow fragment exo- (Kf exo-), calf thymus DNA polymerase alpha (pol alpha) or human DNA polymerase beta (pol beta). Consistent with previous reports, dAMP and dCMP are inserted selectively opposite 8-oxoG with all three DNA polymerases. Interestingly, oxidation of 8-oxoG was found to induce dAMP and dGMP insertion opposite the lesion by Kf exo- with transient inhibition of primer extension occurring at the site of the modified base. Furthermore, the lesion constitutes a block during DNA synthesis by pol alpha and pol beta. Experiments with an 8-oxoA-modified template oligonucleotide show that both 8-oxoA and an oxidized form of 8-oxoA direct insertion of dTMP by Kf exo-. Mass spectrometric analysis of 8-oxoG-containing oligonucleotides before and after oxidation with IrCl62-are consistent with oxidation of primarily the 8-oxoG site, resulting in formation of a guanidinohydantoin moiety as the major product. No evidence for formation of abasic sites was obtained. These results demonstrate that an oxidized form of 8-oxoG, possibly guanidinohydantoin, may direct misreading and misinsertion of dNTPs during DNA synthesis. If such a process occurred in vivo, it would represent a point mutagenic lesion leading to G-->T and G-->C transversions. However, the corresponding oxidized form of 8-oxoA primarily shows correct insertion of T during DNA synthesis with Kf exo-.

Animals↗
PubMed1977

Search for revertants of the glutamine mischarging mutans of Escherichia coli su+3 tyrosine suppressor tRNA that are able to insert tyrosine at the site of amber mutation.

We have isolated a bacterial amber mutation (nadam) that is suppressed by the tyrosine inserting suppressor su+3 but not by the glutamine (su+2, su+3 A1, su+3 G82 and su+3 A1G82), serine (su+1) and leucine (su+6) inserting suppressors. The su+7 suppressor which inserts glutamine and tryptophan also suppresses this mutation indicating that tryptophan, in addition to tyrosine, is accepted at the site of amber mutation. We have used this amber mutation to search for revertants of the su+3 glutamine mischarging mutants su+3 A1, su+3 G82 and su+3 A1G82 that are able to insert tyrosine at the site of amber mutation. Two types of revertants were found in the case of su+3 A1. One type corresponding to the true revertant A1 leads to G, and the other to the second site revertants C81 leads to U (A1U81). The A1U81 revertant has been shown to insert both glutamine and tyrosine at the site of amber mutation. Only true revertants (G82 leads to A) were obtained when su+3 G82 was analyzed. No revertants were obtained in the case of the su+3 A1G82. These results are discussed in relation to aminoacyl-tRNA recognition.

2-Aminopurine↗
PubMed2004

DomIns: a web resource for domain insertions in known protein structures.

Proteins can be formed by single or multiple domains. The process of recombination at the molecular level has generated a wide variety of multi-domain proteins with specific domain organization to cater to the functional requirements of an organism. The functional and structural costs of inserting a domain into another means that multi-domain proteins are usually formed by covalently linking the N-terminus of one domain to the C-terminus of the preceding domain. While this is true in a large proportion of multi-domain proteins, we find a significant fraction of proteins that are the result of domain insertion. The inserted domain breaks the sequence contiguity of the domain into which it is inserted leading to a novel domain organization. This web resource aims to document domain insertions in known protein structures that are classified in the SCOP database. The web server can be accessed from http://stash.mrc-lmb.cam. ac.uk/DomIns/.

Amino Acid Sequence↗
PubMed2005

Gene targeting using randomly inserted group II introns (targetrons) recovered from an Escherichia coli gene disruption library.

The Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse-splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it to prime reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base-pairing of the intron RNA. Consequently, group II introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, we show that an Escherichia coli gene disruption library obtained using such randomly inserting Ll.LtrB introns contains most viable E.coli gene disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. Additionally, we identified a subset of introns that insert at sites lacking T+5, a nucleotide residue critical for second-strand cleavage. All such introns tested individually gave the desired specific disruption, some by switching to an alternate retrohoming mechanism targeting single-stranded DNA and using a nascent lagging DNA strand to prime reverse transcription.

Base Pairing↗
PubMed1997

One-dose cefuroxime i.v. and i.p. reduces microbial growth in PD patients after catheter insertion.

BACKGROUND: When a peritoneal dialysis catheter is inserted intra-abdominally in a patient starting peritoneal dialysis (PD) there is always a risk for postoperative wound infection and peritonitis. At our centre, PD is started immediately after the dialysis catheter is inserted. This may increase the postoperative risk for peritonitis and wound infection. The aim of this prospective, randomized, study was to evaluate whether the incidence of microbial growth postoperatively (within 10 days) after catheter insertion could be reduced by prophylactic antibiotic therapy. SUBJECTS AND METHODS: During a period of 27 months, 38 patients, who consecutively entered the PD programme, (11 women and 27 men, mean age 57 years) were included in the study. Eighteen patients were given cefuroxime 1.5 g i.v. preoperatively and 250 mg i.p. in the first dialysis bag (containing 1 litre fluid) as prophylaxis. Twenty patients were not given prophylactic antibiotics (control group). All catheter insertions were performed in an operating theatre by the same surgeons using the same technique. RESULTS: In the test group, none of the patients showed microbial growth in the dialysis fluid during the post-operative period, while in the control group six of 20 patients (30%) suffered from such growth (P = 0.021). CONCLUSION: Prophylactic treatment by cefuroxime i.v. pre- and i.p. perioperatively may reduce the risk for microbial growth and peritonitis after insertion of a Tenckhoff catheter.

Adult↗
PubMed1998

A hominoid-specific nuclear insertion of the mitochondrial D-loop: implications for reconstructing ancestral mitochondrial sequences.

A nuclear integration of a mitochondrial control region sequence on human chromosome 9 has been isolated. PCR analyses with primers specific for the respective insertion-flanking nuclear regions showed that the insertion took place on the lineage leading to Hominoidea (gibbon, orangutan, gorilla, chimpanzee, and human) after the Old World monkey-Hominoidea split. The sequences of the control region integrations were determined for humans, chimpanzees, gorillas, orangutans, and siamangs. These sequences were then used to construct phylogenetic trees with different methods, relating them with several hominoid, Old Work monkey, and New World monkey mitochondrial control region sequences. Applying maximum-likelihood, neighbor-joining, and parsimony algorithms, the insertion clade was attached to the branch leading to the hominoid mitochondrial sequences as expected from the PCR-determined presence/absence of this integration. An unexpected long branch leading to the internal node that connects all insertion sequences was observed for the different phylogeny reconstruction procedures. This finding is not totally compatible with the lower evolutionary rate in the nucleus than in the mitochondrial compartment. We determined the unambiguous substitutions on the branch leading to the most recent common ancestor (MRCA) of the mitochondrial inserts according to the parsimony criterium. We propose that they are unlikely to have been caused by damage of the transposing nucleic acid and that they are probably due to a change in the evolutionary mode after the transposition.

Animals↗
PubMed1989

Structure-function analysis of tissue-type plasminogen activator by linker-insertion, point and deletion mutagenesis.

We undertook a structure--function analysis of human tissue plasminogen activator (tPA) using linker-scanning and deletion mutagenesis. Synthetic oligonucleotide linkers were introduced into the tPA cDNA at pre-existing restriction enzyme sites. This generated a series of tPA variants which contained small primary sequence alterations consisting of point mutations, deletions or insertions. The majority of the linker-insertion variants demonstrate a significant reduction in amidolytic and fibrinolytic activity in comparison to wild-type tPA. The exceptions are the variants with linker-inserts placed at the BglII(115) and StyI(277) sites of the tPA cDNA (4SLEG5 and 57LEA58 respectively), which encode insertions at the boundaries of the finger domain. The variants with linker-inserts in the light chain (protease domain) of tPA are the lowest in enzymatic activity. Particularly sensitive to mutation are highly conserved amino acids. Heavy chain deletion variants were constructed from point mutants at the domain boundaries of tPA. Deletion of the kringle domains lowers the fibrinolytic activity to a greater extent than deletion of the finger or growth factor domains. We conclude that alterations in any domain of the tPA molecule, and particularly in the highly conserved residues within these domains, can affect fibrinolytic activity.

Amino Acid Sequence↗
PubMed2005

Integrity of nonviral fragments in recombinant Tomato bushy stunt virus and defective interfering RNA is influenced by silencing and the type of inserts.

Recombinant plant viruses have the propensity to remove foreign inserts during replication. This process is virus-specific and occurs in a host-dependent manner. In the present study, we investigated the integrity of foreign inserts in recombinant plant viruses using a model system consisting of Tomato bushy stunt virus (TBSV) and its defective interfering RNA (DI). These were tested in Nicotiana benthamiana plants that were either wild type or transgenic for the green fluorescent protein (GFP) gene. GFP-derived inserts were retained in the recombinant TBSV and DI population that were inoculated onto GFP-transgenic N. benthamiana plants in which silencing of the GFP transgene was initiated, but they were removed from the virus and DIs that were maintained on wild-type plants. A foreign insert derived from an endogenous N. benthamiana gene encoding the H subunit of the magnesium chelatase (NbChlH) was deleted, whereas the fragment of an RNA-dependent RNA polymerase gene (NbRdRP1m) was retained in the recombinant TBSV population. These results demonstrate that the recombination of TBSV to remove nonviral fragments is influenced by silencing and the type of inserts.

DNA, Recombinant↗
PubMed1990

Acute modification of biomechanical properties of the bone-ligament insertion to rat limb unweighting.

We investigated the acute adaptation of the rat femur-medial collateral ligament-tibia (FMT) complex to 7 days of limb unweighting by means of a hind-limb suspension protocol. Male, young adult, Harlan Sprague-Dawley rats were randomly assigned to either control or suspended groups. Rats deprived of hind limb-to-ground contact forces had a 42% decrease in soleus muscle mass compared with the control group. Medial collateral ligament (MCL) length and cross-sectional area were measured, and each FMT complex was tension tested to failure. All failed at their tibia-MCL insertion. The ultimate load in the FMT and the peak Kirchhoff stress in the MCL (occurring immediately before insertion site failure) were significantly reduced in the suspended group. The suspended MCLs were 9.7% larger in area and 5.7% shorter in length than the controls under the same preload (0.25 N). We found no significant differences between the control and suspended MCLs in Green strain, stretch, or deformation immediately before insertion site failure, nor did we find a significant difference in the MCL tangent modulus. This study indicates that even acute periods of limb unweighting can structurally compromise bone-ligament insertions. Further, this study implies that the adaptations responsible for this structural compromise must involve acute changes in the intrinsic zone (or zones) of the bone-ligament insertion.

Animals↗
PubMed1997

The effect of skull-pin insertion on cerebrospinal fluid pressure and cerebral perfusion pressure: influence of sufentanil and fentanyl.

This randomized prospective study measured the effects of an intravenous opioid bolus on cerebrospinal fluid pressure (CSFP), mean arterial pressure (MAP), and cerebral perfusion pressure (CPP) during skull-pin insertion. Twenty-two adult patients scheduled for elective craniotomy for supratentorial lesions were studied. Outcome variables were MAP, heart rate (HR), and lumbar CSFP. The standardized anesthetic regimen included fentanyl (2 microg/kg), thiopental (5-7 mg/kg), lidocaine (1.5 mg/kg), isoflurane (0.3-0.7 minimum alveolar anesthetic concentration), and vecuronium (0.1 mg/kg). During stable anesthesia, sufentanil (0.8 microg/kg) or fentanyl (4.5 microg/kg) was given as a bolus before skull-pin insertion. The hemodynamic effects of the opioid injection were modified with phenylephrine and/or atropine when indicated. CSFP remained unchanged in both treatment groups. MAP and CPP increased approximately 10 mm Hg after skull-pin insertion (P<0.001). In the sufentanil group, HR decreased approximately 10 bpm after opioid injection and remained decreased throughout the study. In fentanyl-treated patients, HR decreased 8 bpm after opioid injection but returned to preopioid rates after skull-pin insertion. In conclusion, in anesthetized patients, an intravenous bolus of fentanyl or sufentanil prior to skull-pin insertion results in stable values of CSFP, CPP, BP, and HR when the hemodynamic effects of the opioid are modified with phenylephrine and atropine.

Adjuvants, Anesthesia↗
PubMed2002

A comparison of ketamine and lidocaine spray with propofol for the insertion of laryngeal mask airway in children: a double-blinded randomized trial.

UNLABELLED: The laryngeal mask airway (LMA) has been used successfully as both a ventilatory device and a conduit for tracheal intubation. In this double-blinded, randomized study, we examined whether pretreatment with lidocaine spray, ketamine anesthesia, and LMA insertion could be used as airway management that could maintain spontaneous breathing in children. After IV premedication with midazolam 0.05 mg/kg and glycopyrrolate 0.005 mg/kg, 90 patients were randomly allocated to 1 of 2 main groups for the administration of either propofol or ketamine: 40 patients received 2.5, 3.0, 3.5, or 4.0 mg/kg of propofol IV (n = 10 each), whereas 50 patients received 2.0, 2.5, 3.0, 3.5, or 4.0 mg/kg of ketamine IV (n = 10 each). Only in the ketamine group was lidocaine spray applied to the oropharynx 1 min before anesthesia induction. After injection of the designated drug, self-respiration, airway obstruction, and jaw relaxation were checked. Self-respiration, laryngospasm coughing, gagging, swallowing, biting or tongue movements, secretions, and head or limb movements after LMA insertion were graded. All variables were graded as satisfactory, acceptable, or unsatisfactory. The overall result was considered satisfactory if all criteria were satisfactory; acceptable if all were better than acceptable, but at least one acceptable criterion was included; and unsatisfactory if at least one criterion was unsatisfactory. Overall satisfactory or acceptable results in every patient were achieved only in the ketamine 3.0 or 3.5 mg/kg subgroups. No propofol dose was completely satisfactory; most cases involved apnea or airway obstruction. Ketamine and lidocaine spray were appropriate for LMA insertion, which may be a safe method for management of difficult airway in children. IMPLICATIONS: Ketamine and lidocaine spray appear to be appropriate for laryngeal mask airway (LMA) insertion in children. Thus, apnea and airway obstruction, the two most serious and frequent complications of propofol, can be avoided during LMA insertion.

Child↗
PubMed2002

Coventry Award paper. Backsurface wear and deformation in polyethylene tibial inserts retrieved postmortem.

Wear and deformation were characterized at the backsurface of 25 posterior cruciate-retaining total knee arthroplasty polyethylene inserts retrieved postmortem from 20 subjects. The mean implantation time was 64.1 months (range, 4-156 months). The backsurface of the inserts was inspected using a stereomicroscope with a digital optical system. Coronal histologic sections of 13 proximal tibias were inspected for the presence and extent of penetration of granuloma. Damage to the backsurface was limited. Polishing was recorded on 21 (84%) of the inserts and abrasive wear on five (20%) inserts. Pitting was present in 21 (84%) components, but involved less than 1% of the area in all but one of these components. Delamination and cracking were not observed. Extrusions were seen in all 10 of the components that had screw holes in the tibial tray. A correlation was found between the depth of penetration of the granuloma along the posteromedial screw and the height of the corresponding extrusion. The anteroposterior profiles showed a concave deformation of the backsurface in 24 (96 %) of the cases. The concave deformation of tibial inserts may facilitate accumulation and transportation of wear debris to the tibial bone-implant interface through the screw holes in implants designed for cementless fixation.

Aged↗
PubMed1985

Forward displacement of the larynx for nasogastric tube insertion in intubated patients.

Simple insertion of a nasogastric (NG) tube was successful in only 52 of 100 anesthetized intubated patients. After the larynx was manually pulled forward, the NG tube was successfully inserted in 33 patients. In the remaining 15 patients, the NG tube was inserted only with the aid of a finger or a laryngoscope used with Magill forceps. The difficulty of NG tube insertion was not correlated with sex, age, weight, or type of endotracheal intubation. Forward displacement of the larynx by manually gripping and lifting the thyroid cartilage is a useful and safe maneuver that facilitates NG tube insertion in anesthetized intubated patients.

Adolescent↗