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PubMed1992

Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes.

An insertion of about 100 bases within the central part of the 23S rRNA genes was found to be a phylogenetic marker for the bacterial line of descent of Gram-positive bacteria with a high DNA G + C content. The insertion was present in 23S rRNA genes of 64 strains representing the major phylogenetic groups of Gram-positive bacteria with a high DNA G+C content, whereas it was not found in 23S rRNA genes of 55 (eu)bacteria representing Gram-positive bacteria with a low DNA G + C content and all other known (eu)bacterial phyla. The presence of the insertion could be easily demonstrated by comparative gel electrophoretic analysis of in vitro-amplified 23S rDNA fragments, which contained the insertion. The nucleotide sequences of the amplified fragments were determined and sequence similarities of at least 44% were found. The overall similarity values are lower than those of 16S and 23S rRNA sequences of the particular organism. Northern hybridization experiments indicated the presence of the insertion within the mature 23S rRNA of Corynebacterium glutamicum.

Base Composition↗
PubMed1997

A new insertion sequence IS1452 from Acetobacter pasteurianus.

A new insertion sequence element, IS1452, was found to be associated with inactivation of the alcohol dehydrogenase by insertion in the adhS gene encoding subunit III of the three-component membrane-bound alcohol dehydrogenase complex in Acetobacter pasteurianus. Cloning and sequencing analyses of the mutated subunit III gene locus revealed that IS1452 was inserted at or near the ribosome-binding sequence of adhS. Analysis of transcription using the chloramphenicol acetyltransferase gene as the reporter indicated that IS1452 abolished transcription of adhS by separating its promoter from the subunit III structural gene. IS1452 was 1411 bp in length and had a terminal inverted repeat of 21 bp. IS1452 contained one long ORF of 416 amino acids rich in basic amino acids. This protein showed homology with a putative transposes, Tra1, of IS701 isolated from the cyanobacterium Calothrix species PCC 7601. Like IS701, IS1452 was found to generate a 4 bp direct repeat at the site of insertion upon transposition. The target site specificity was rather strict, and a CTA(A or G) sequence appeared to be preferentially recognized. Transposition of IS1452 was replicative, since it was accompanied by an increase in the copy number of IS1452. Several strains belonging to the genus Acetobacter also contained IS1452 at varying copy numbers from one to more than ten. These observations suggest that IS1452 is one of the insertion sequences that are responsible for genetic instability leading to deficiencies in various physiological properties in acetic acid bacteria.

Acetobacter↗
PubMed1999

IS1626, a new IS900-related Mycobacterium avium insertion sequence.

An insertion sequence designated IS1626 was isolated and characterized from a Mycobacterium avium clinical strain. IS1626 was detected by high-stringency hybridization with the pMB22/S12 probe from IS900 of Mycobacterium paratuberculosis. IS1626 is 1418 bp in size and has a G+C content of 65 mol%. It has neither terminal inverted repeats nor flanking direct repeats. Analysis of three IS1626 insertion sites in the M. avium strain and the corresponding potential insertion sites in two IS1626-free M. avium strains indicated a consensus sequence of CATGCN(4-5)TCCTN(2)G for IS1626 insertion. In the three clones examined, IS1626 has the same orientation with respect to this target site. IS1626 has two major ORFs. ORF1179 encodes a predicted protein of 393 amino acids. ORF930, on the complementary strand of ORF1179, encodes a protein of 310 amino acids. The Shine-Dalgarno sequence for ORF930 is partially located in the flanking region, similar to other IS900-related elements. Analysis of the comparable features of insertion sequences and their variable occurrence in related organisms is useful for studying the evolution of these elements and their hosts.

Amino Acid Sequence↗
PubMed2006

Influence of naturally occurring insertions in the fingers subdomain of human immunodeficiency virus type 1 reverse transcriptase on polymerase fidelity and mutation frequencies in vitro.

The fingers subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a hotspot for nucleoside analogue resistance mutations. Some multi-nucleoside analogue-resistant variants contain a T69S substitution along with dipeptide insertions between residues 69 and 70. This set of mutations usually co-exists with classic zidovudine-resistance mutations (e.g. M41L and T215Y) or an A62V mutation and confers resistance to multiple nucleoside analogue inhibitors. As insertions lie in the vicinity of the dNTP-binding pocket, their influence on RT fidelity was investigated. Commonly occurring insertion mutations were selected, i.e. T69S-AG, T69S-SG and T69S-SS alone, in combination with 3'-azido-2',3'-deoxythymidine-resistance mutations M41L, L210W, R211K, L214F, T215Y (LAG(AZ) and LSG(AZ)) or with an alternate set where A62V substitution replaces M41L (VAG(AZ), VSG(AZ) and VSS(AZ)). Using a lacZalpha gapped duplex substrate, the forward mutation frequencies of recombinant wild-type and mutant RTs bearing each of the above sets of mutations were measured. All of the mutants displayed significant decreases in mutation frequencies. Whereas the dipeptide insertions alone showed the least decrease (4.0- to 7.5-fold), the VAG series showed an intermediate reduction (5.0- to 11.4-fold) and the LAG set showed the largest reduction in mutation frequencies (15.3- and 16.3-fold for LAG(AZ) and LSG(AZ), respectively). Single dNTP exclusion assays for mutants LSG(AZ) and LAG(AZ) confirmed their large reduction in misincorporation efficiencies. The increased in vitro fidelity was not due to excision of the incorrect nucleotide via ATP-dependent removal. There was also no direct correlation between increased fidelity and template-primer affinity, suggesting a change in the active site that is conducive to better discrimination during dNTP insertion.

Amino Acid Substitution↗
PubMed1991

legless insertional mutation: morphological, molecular, and genetic characterization.

Limb morphogenesis is an excellent model system to study pattern formation during vertebrate development. The legless (lgl) insertional mutation can serve as a tool to analyze specific events in limb development at the embryologic, genetic, and molecular levels. Hemizygous mice of this transgenic line are phenotypically normal, but homozygous mutants are inviable and exhibit limb, brain, and craniofacial malformations, as well as situs inversus. By morphological analysis of mutant hindlimb buds we show absence of a normal apical ectodermal ridge, a structure required for limb bud outgrowth, and an unusually high degree of mesenchymal and ectodermal cell death. Mutant embryos are extremely sensitive to retinoic acid, a known teratogen with a proposed role in limb development. The hindlimb malformations in legless mutants are less severe when bred into the BALB/c background, suggesting the involvement of other strain-specific genes. Molecular analysis of the disrupted region indicates two tightly linked insertion sites. Sequences flanking the transgene insertions have been cloned and mapped to chromosome 12, near the iv (situs inversus viscerum) locus. Consistent with this, complementation tests confirm allelism of lgl and iv and suggest that the transgene insertion may have disrupted more than one gene. Phylogenetically conserved sequences flanking the transgene insertions were identified and used to isolate candidate lgl and iv cDNAs.

Alleles↗
PubMed1995

Light-harvesting chlorophyll a/b-binding protein inserted into isolated thylakoids binds pigments and is assembled into trimeric light-harvesting complex.

The light-harvesting chlorophyll a/b-binding protein (LHCP) is largely protected against protease (except for about 1 kD on the N terminus) in the thylakoid membrane; this protease resistance is often used to assay successful insertion of LHCP into isolated thylakoids in vitro. In this paper we show that this protease resistance is exhibited by trimeric light-harvesting complex of photosystem II (LHCII) but not by monomeric LHCII in which about 5 kD on the N terminus of LHCP are cleaved off by protease. When a mutant version of LHCP that is unable to trimerize in an in vitro reconstitution assay is inserted into isolated thylakoids, it gives rise to only the shorter protease digestion product indicative of monomeric LHCII. We conclude that more of the N-terminal domain of LHCP is shielded in trimeric than in monomeric LHCII and that this difference in protease sensitivity can be used to distinguish between LHCP assembled in LHCII monomers or trimers. The data presented prove that upon insertion of LHCP into isolated thylakoids at least part of the protein spontaneously binds pigments to form LHCII, which then is assembled in trimers. The dependence of the protease sensitivity of thylakoid-inserted LHCP on the oligomerization state of the newly formed LHCII justifies caution when using a protease assay to verify successful insertion of LHCP into the membrane.

Amino Acid Sequence↗
PubMed1992

Alternative splicing induced by insertion of retrotransposons into the maize waxy gene.

The molecular basis for the low level expression of three alleles of the maize waxy (Wx) gene has been described. Each allele contains a retrotransposon in intron sequences. These insertions represent previously undescribed elements, and their association with three wx alleles indicates that retrotransposon elements are important agents of spontaneous mutation in maize. For each allele, element sequences are spliced from pre-mRNA with the surrounding intron even though the insertions increase intron length by approximately 40- to 60-fold. In addition, despite differences in element sequences, insertion sites, and relative orientations, each element disrupts long-range splice site recognition leading to novel Wx transcripts where exons both upstream and downstream of the insertion site are skipped. The expression of wx alleles with large insertions in introns provides support for studies that indicate that the primary cis requirement for maize introns is the splice donor and acceptor sites.

Alternative Splicing↗
PubMed1993

The peri-operative management of surgical insertion and removal of the intravenous oxygenator device (IVOX). A report of nine cases.

Intravenous oxygenation (IVOX) is a new technique for augmentation of gas exchange in patients who require near maximal conventional ventilatory support. Patients who require IVOX are, by definition, critically ill with a high expected mortality. At present, these high risk patients must be transferred to the operating theatre for the IVOX device to be inserted. This report describes the anaesthetic problems associated with nine patients in whom an IVOX device was inserted and removed in our institution. The mortality was six out of nine patients; all deaths occurred with the IVOX device in situ. Three patients died within 6 h of insertion. Four patients were female. The patients' ages ranged from 14 to 76 years. There were few immediate ventilation changes in the first 4 h after IVOX insertion. Inspired oxygenation concentration was reduced in only one patient. Positive end-expiratory pressure was not reduced. Peak inspiratory pressure decreased in four patients. Arterial oxygen tension increased in four patients (range 0.1-2.5 kPa) and decreased in five (range 0.1-3.4 kPa). Arterial carbon dioxide tension increased in one patient (0.3 kPa) and decreased in eight (range 0.1-2.7 kPa). Inotropic support with adrenaline, dobutamine and noradrenaline needed to be initiated or increased in eight patients. Eight patients required 2-4 units of blood to be transfused during IVOX insertion or in the following 2 h. One patient suffered an asystolic cardiac arrest during the operation, but was resuscitated successfully. Three patients survived to have the IVOX removed.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

A comparison of arterial lines and insertion techniques in critically ill patients.

We compared three arterial line insertion techniques and two types of arterial catheters in 69 critically ill patients. Use of the direct-puncture technique (method A) was associated with a significantly higher failure rate (23%) than use of a catheter with a separate guide wire (method B, 'classical' Seldinger technique, p < 0.001) or a catheter with an integral guide wire (method C, 'modified' Seldinger technique, p < 0.02). Operators randomly allocated to using method A took significantly longer to perform the procedure than those using method C (p < 0.01), used significantly more catheters (p < 0.0001) and made significantly more punctures in achieving a successful insertion than those using either methods B (p < 0.001) or C (p < 0.001). Both catheter types B and C (polyurethane) were significantly less likely to block, thus requiring less likely to block, thus requiring re-insertion, than catheter type A (Teflon) (p < 0.02, p < 0.01 respectively). We recommend the use of a 'classical' Seldinger technique (method B) for arterial line insertion in critically ill patients and the use of a polyurethane catheter, in preference to Teflon, to maximise catheter life after insertion.

Adolescent↗
PubMed1995

Facilitation of laryngeal mask insertion. Effects of lignocaine given intravenously before induction with propofol.

The effects of pretreatment with lignocaine administered intravenously on the insertion of the laryngeal mask airway were investigated in 80 unpremedicated, ASA 1 or 2, adult day-case patients in a randomised, double-blind, placebo-controlled trial. Patients received either intravenous lignocaine 1.5 mg.kg-1 or an equivalent volume of sodium chloride 0.9%. Induction of anaesthesia was achieved with propofol given via a syringe driver at a fixed rate of 600 ml.h-1 until the patient dropped a weighted syringe. No opioid or sedative drugs were given prior to induction. Pain on injection of propofol was recorded. Jaw opening, ease of insertion of the laryngeal mask, coughing, gagging and airway patency were all scored on three-point scales immediately after mask insertion re-attempted. This cycle was continued until success was achieved and the number of such cycles recorded. There were no differences between the lignocaine and control groups with respect to induction dose of propofol, degree of jaw opening, or amount of gagging. Laryngeal mask insertion was facilitated by pretreatment with lignocaine administered intravenously, without an alteration in induction dose of propofol (p < 0.05). Coughing and airway obstruction were both significantly reduced by pretreatment with lignocaine, as was the incidence of failure of insertion requiring deepening of anaesthesia (p < 0.05).

Adult↗
PubMed1996

Lignocaine to aid the insertion of the laryngeal mask airway with thiopentone. A comparison between topical and intravenous administration.

Conditions for insertion of a laryngeal mask airway in 90 unpremedicated adult were patients were assessed in a randomised, single-blinded trial. Each patient received fentanyl 1 microgram.kg-1 and thiopentone 5 mg.kg-1, and this was preceded either by lignocaine 0.5 mg.kg-1 intravenously (group 1), lignocaine 1.5 mg.kg-1 intravenously (group 2) or 40 mg of topical lignocaine spray to the posterior pharyngeal wall (group 3). Conditions for laryngeal mask airway insertion were recorded. The group receiving topical lignocaine had a lower incidence of laryngospasm (p < 0.05), required fewer attempts for successful insertion of the laryngeal mask (p < 0.05) and coughed or gagged less frequently than either group receiving lignocaine intravenously (p > 0.05). Overall, the conditions for laryngeal mask airway insertion were better in the topical group (p < 0.05). There were no significant differences in haemodynamic response and apnoea between the three groups. Topical lignocaine spray prior to thiopentone provides conditions for insertion of a laryngeal mask that are superior to those provided by lignocaine and thiopentone intravenously.

Administration, Topical↗
PubMed1996

Insertion methods of the laryngeal mask airway. A survey of current practice in Wales.

The efficacy of the laryngeal mask is widely accepted, but there is a lack of consensus on the best insertion method and on the use of the mask for certain surgical procedures. We sent a questionnaire to all anaesthetists in Wales to discover the frequency of use of the laryngeal mask and the preferred insertion method. The questionnaire also enquired about the use of the laryngeal mask during anaesthesia for laparoscopic clip sterilisation. Replies were received from 125 consultants (89% of those circulated) and 122 non-consultants (69%). The insertion method described in the manufacturer's instruction manual was preferred by 30% of consultants and 34% of the others. The next most popular option was insertion of the mask with the cuff partially inflated. Twenty-three per cent of consultants and 34% of non-consultants were prepared to use the laryngeal mask during anaesthesia for laparoscopic clip sterilisation. Although the insertion technique described in the instruction manual is the most widely employed, a large number of alternative methods are frequently used.

Anesthesia↗
PubMed1997

Ease of insertion of the laryngeal mask airway by inexperienced personnel when using an introducer.

The Portex introducer for the laryngeal mask airway was designed as an aid to successful insertion, acting as an idealised 'artificial hard palate' to guide the tip of the laryngeal mask into the correct position. A number of authors have investigated laryngeal mask insertion by unskilled personnel in certain situations, one example being nurses during in-hospital cardiopulmonary resuscitation. We investigated whether the introducer had any effect on the incidence of first-time successful LMA placement by unskilled personnel. These were nonanaesthetist doctors, randomly assigned to have one attempt at LMA insertion in an anaesthetised patient, with and without the introducer. In 44 patients with the LMA being inserted according to the manufacturer's instructions, there was a 68% success rate (14 failures). In 45 patients with the LMA being inserted with the aid of a Portex introducer, there was a 96% success rate (two failures). This was a highly significant improvement (p < 0.001).

Adolescent↗
PubMed1997

Intra-ocular pressure changes following laryngeal mask airway insertion: a comparative study.

We compared the effects of the Brain laryngeal mask airway with a tracheal tube on intra-ocular pressure. Propofol was used as induction agent and atracurium as relaxant. Twenty-six patients with normal intra-ocular pressure undergoing cataract surgery were randomly allocated to two groups. Group A (n = 13) had a laryngeal mask airway inserted and Group B (n = 13) had a tracheal tube inserted. Intra-ocular pressure was measured just before insertion of the airway, 20 s after insertion and at 2 min. In the laryngeal mask airway group there were no significant changes in mean intra-ocular pressure. In the tracheal tube group there was a significant rise in mean intra-ocular pressure at 20 s (p = 0.0056) which returned to pre-insertion levels at 2 min. We conclude that the laryngeal mask airway continues to have advantages over the tracheal tube for ophthalmic surgery despite the use of propofol and atracurium as anaesthetic agents.

Aged↗
PubMed1990

Insertion of an extra amino acid is the main cause of the low affinity of penicillin-binding protein 2 in penicillin-resistant strains of Neisseria gonorrhoeae.

Non-beta-lactamase-producing, penicillin-resistant strains of Neisseria gonorrhoeae (CMRNG strains) produce altered forms of penicillin-binding protein 2 (PBP2) that have decreased affinity for penicillin. A feature of PBP2 from all CMRNG strains is the presence of an additional residue (Asp-345A) that is absent from PBP2 of penicillin-sensitive strains. The role of the additional aspartic acid residue in the decreased affinity of PBP2 is unclear as PBP2 of all previously examined CMRNG strains possess several other amino acid sequence alterations, in addition to the insertion of Asp-345A, compared to PBP2 of penicillin-sensitive strains. Site-directed mutagenesis has been used to insert the Asp-345A codon into the penA gene from a penicillin-sensitive gonococcus. The resulting penA gene expressed an altered form of PBP2 that had a decreased affinity for benzylpenicillin and was able to transform a penicillin-sensitive strain of N. gonorrhoeae to an increased level of resistance to benzylpenicillin. Insertion of amino acids other than aspartic acid did not produce forms of PBP2 that provided increased resistance to penicillin. Removal of the Asp-345A codon from the penA gene of a CMRNG strain reduced its ability to transform a penicillin-sensitive strain to an increased level of penicillin resistance. The reduction in the affinity of PBP2 in CMRNG strains is therefore largely, although not exclusively, due to the insertion of Asp-345A. Clinical isolates that produce altered forms of PBP2 that differ from that of penicillin-sensitive strains only in the insertion of Asp-345A have been identified.

Amino Acid Sequence↗
PubMed1991

Characterization of two insertion sequences, IS701 and IS702, from the cyanobacterium Calothrix species PCC 7601.

We describe the characterization of two insertion elements, IS701 and IS702, isolated from Calothrix species PCC 7601. These insertion elements were cloned from spontaneous pigmentation mutants. Both show the characteristics of typical bacterial insertion sequences, i.e. they present long terminal inverted repeats and they duplicate target DNA upon insertion. These elements share no homology with the only other cyanobacterial insertion sequence described so far, IS891. At least 15 copies of IS701 and 9 copies of IS702 were detected by hybridization experiments in the Calothrix 7601 genome. Their occurrence in several cyanobacterial strains is also reported.

Amino Acid Sequence↗
PubMed1995

The somatic hypermutation activity of a follicular lymphoma links to large insertions and deletions of immunoglobulin genes.

A biopsy specimen from a patient with follicular lymphoma was divided into two fragments. DNA was extracted from one fragment and a 1.2 kb region of the functional heavy chain (IgH) gene was amplified, cloned and sequenced (eight clones). From the other fragment a cell line (HF-1) was started. The IgH gene region was amplified from the cell line, and sequenced without cloning. The nine sequences obtained could be arranged into a genealogical tree where the individual sequences differed from the deduced ancestor by 16-29 single nucleotide changes, some also by an insertion and/or a deletion. It is apparent that the sequence alterations were caused by somatic mutations during the growth of the lymphoma. The comparison of the sequences with two published (allelic) germline sequences of the human JH region showed approximately 20% non-homology. The differences included five additional multinucleotide insertion/deletion changes, the longest of them a 101-nucleotide insertion. Two long insertions were homologous to the adjacent germline sequences. We propose that most of the changes observed, including long deletions and insertions, represent or are linked to somatic hypermutation events of the Ig gene type. Although in a few cases large deletions and insertions (> 2 bp) have been found in mutated immunoglobulin genes, our results, for the first time, firmly link these deletions/insertions to somatic hypermutations; their frequency was found to be 2.2% of the observed mutational events in the non-translated gene regions. HF-1 is the first follicular lymphoma line successfully established from a lymphoma known to have hypermutated its Ig genes during the malignant growth. It is a candidate cell line to be studied for its ability to generate mutations of B cell type in cell cultures.

Base Sequence↗
PubMed1981

Synthesis and insertion, both in vivo and in vitro, of rat-liver cytochrome P-450 and epoxide hydratase into Xenopus laevis membranes.

We described whole cell and cell-free systems capable of inserting into membranes cytochrome P-450 and epoxide hydratase made under the direction of rat liver RNA. The systems have been used to study the pathways followed by newly made secretory and integral membrane proteins. The cell-free system contains Xenopus laevis embryo membranes, and demonstrates competition for a common receptor between cytochrome P-450 and epoxide hydratase, and normal secretory proteins: evidence is provided for differential membrane receptor affinity. Thus, synthesis of secretory and membrane proteins appears to involve a common initial pathway. Microinjection of rat liver RNA into whole oocytes suggests that membrane insertion is neither cell type nor species specific, because functional rat liver enzymes are found inserted in the endoplasmic reticulum of the frog cell. Nonetheless, insertion is highly selective since albumin and several other proteins made under the direction of the injected liver RNA are sequestered within membrane vesicles and are then secreted by the oocyte, whilst epoxide hydratase and cytochrome P-450 are inserted into membranes but are not secreted.

Animals↗