SEARCH · PubMed Health

Results for “insertion”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources

PubMed2005

Molecular analysis of Arabidopsis endosperm and embryo promoter trap lines: reporter-gene expression can result from T-DNA insertions in antisense orientation, in introns and in intergenic regions, in addition to sense insertion at the 5' end of genes.

Random insertions of promoterless reporter genes in genomes are a common tool for identifying marker lines with tissue-specific expression patterns. Such lines are assumed to reflect the activity of endogenous promoters and should facilitate the cloning of genes expressed in the corresponding tissues. To identify genes active in seed organs, plant DNA flanking T-DNA insertions (T-DNAs) have been cloned in 16 Arabidopsis thaliana GUS-reporter lines. T-DNAs were found in proximal promoter regions, 5' UTR or intron with GUS in the same (sense) orientation as the tagged gene, but contrary to expectations also in inverted orientation in the 5' end of genes or in intergenic regions. RT-PCR, northern analysis, and data on expression patterns of tagged genes, compared with the expression pattern of the reporter lines, suggest that the expression pattern of a reporter gene will reflect the pattern of a tagged gene when inserted in sense orientation in the 5' UTR or intron. When inserted in the promoter region, the reporter-gene expression patterns may be restricted compared with the endogenous gene. Among the trapped genes, the previously described nitrate transporter gene AtNRT1.1, the cyclophilin gene ROC3, and the histone deacetylase gene AtHD2C were found. Reporter-gene expression when positioned in antisense orientation, for example, in the SLEEPY1 gene, is indicative of antisense expression of the tagged gene. For T-DNAs found in intergenic regions, it is suggested that the reporter gene is transcribed from cryptic promoters or promoters of as yet unannotated genes.

Arabidopsis↗

PubMed1989

Insertional pain and other IUD insertion-related rare events for breastfeeding and non-breastfeeding women--a decade's experience in developing countries.

The possible effect of breastfeeding on intrauterine device (IUD) insertion events was investigated. Analysis included a total of 6493 women who enrolled in multicenter IUD clinical trials over a ten-year period. Findings indicate that breastfeeding exerts a protective effect on the incidence of moderate to severe insertional pain and reduces the need for cervical dilatation to facilitate insertion. The pain protection effect was most evident in breastfeeding women who were still in lactational amenorrhea. Subjects with amenorrhea, both breastfeeding and non-breastfeeding, had a significantly lower incidence of pain at IUD insertion than the corresponding menstruating subjects. This effect may be related to a higher secretion of beta-endorphin in the breastfeeding and lactational amenorrheic subjects.

Breast Feeding↗

PubMed1991

Vectors for inserting selectable markers in vector arms and human DNA inserts of yeast artificial chromosomes (YACs).

To facilitate studies of gene expression and homologous recombination, plasmids have been developed which permit the insertion of neomycin resistance-encoding gene (NmR) into either the human DNA insert or the vector arm of a yeast artificial chromosome (YAC). To integrate into the YAC arm, the plasmid pRV1 contains a LYS2 (encoding alpha-aminoadipate reductase) gene for selection in the yeast host, and a NmR gene for subsequent selection after transfection of mammalian cells. These two sequences are bracketed by fragments of the URA3 gene (encoding orotidine-5'-phosphate decarboxylase) that can disrupt the URA3 gene in the YAC arm by homologous recombination in yeast. To integrate a selectable marker into the insert, the plasmid pRV2 contains a NmR gene and an intact copy of the URA3 gene, bracketed by segments of an L1 (LINEs) repetitive element. In this case, the vector has been designed for use with YACs that have already been fitted in the vector arm with a different marker (i.e., TK) that has disrupted the URA3 gene in the vector arm. Selection is for the restoration of URA3 gene activity attendant on recombination into an L1 element in the YAC insert. Use of the vectors is illustrated with a YAC clone containing ribosomal DNA.

Aldehyde Oxidoreductases↗

PubMed2003

Follow-up visits after IUD-insertion: sense or nonsense? A technology assessment study to analyze the effectiveness of follow-up visits after IUD insertion.

The objective of this study is to evaluate whether regular follow-up after insertion of an IUD protects against the risk and side effects of this contraceptive device. To study the effectiveness of the follow-up visits done after IUD insertion, we compared a group of women with regular follow-up visits (group A: after 6 weeks, 3, 6 and 12 months) with women who had non-regular follow-up visits (group B: after 6 weeks and annually) for pregnancy, expulsion rates and discontinuation. A total of 280 women were included (group A: 199, group B: 81). Three pregnancies and 2 unnoticed expulsions were observed. The detection rate of an unnoticed expulsion is 0.35 and 0.11 per 100 visits for, respectively, the visit 6 weeks after insertion and all successive visits. Women in group A came significantly more frequently for unscheduled visits [relative risk (RR): 1.6; 95% confidence interval (CI): 1.03-2.4]. The number of discontinuations, pregnancies and expulsions did not differ between groups, but women in group B had their IUD removed earlier (p < 0.01). We conclude that regular follow-up after the insertion of an IUD is not effective.

Adult↗

PubMed1997

Molecular cloning of cDNA encoding the c-kit receptor of Shiba goats and a novel alanine insertion specific to goats and sheep in the kinase insert region.

The complete open reading frame (ORF) of the c-kit cDNA was cloned from a cerebellar cDNA library of the Shiba goat (Capra hircus var Shiba) with the dominant black-eyed white phenotype. The analysis of the deduced amino acid sequence revealed the presence of a single amino acid insertion (alanine) in the kinase insert (KI) region. While the newly found alanine insertion is not correlated with the coat color phenotype of goats, it appears to be characteristic of the c-kit genes in goats and sheep. Although the biological significance of the insert remains to be investigated, its phylogenetically limited distribution will provide us with a useful and interesting tool to analyze the problems of evolution of sheep and goats in bovidae.

Alanine↗

PubMed1999

A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256.

Biofilm formation of Staphylococcus epidermidis on smooth polymer surfaces has been shown to be mediated by the ica operon. Upon activation of this operon, a polysaccharide intercellular adhesin (PIA) is synthesized that supports bacterial cell-to-cell contacts and triggers the production of thick, multilayered biofilms. Thus, the ica gene cluster represents a genetic determinant that significantly contributes to the virulence of specific Staphylococcus epidermidis strains. PIA synthesis has been reported recently to undergo a phase variation process. In this study, biofilm-forming Staphylococcus epidermidis strains and their PIA-negative phase variants were analysed genetically to investigate the molecular mechanisms of phase variation. We have characterized biofilm-negative variants by Southern hybridization with ica-specific probes, polymerase chain reaction and nucleotide sequencing. The data obtained in these analyses suggested that in approximately 30% of the variants the missing biofilm formation was due to the inactivation of either the icaA or the icaC gene by the insertion of the insertion sequence element IS256. Furthermore, it was shown that the transposition of IS256 into the ica operon is a reversible process. After repeated passages of the PIA-negative insertional mutants, the biofilm-forming phenotype could be restored. Nucleotide sequence analyses of the revertants confirmed the complete excision of IS256, including the initially duplicated 8 bp target sites. These results elucidate, for the first time, a molecular mechanism mediating phase variation in staphylcocci, and they demonstrate that a naturally occurring insertion sequence element is actively involved in the modulation of expression of a Staphylococcus virulence factor.

Bacterial Adhesion↗

PubMed1988

Specific insertion and deletion of insertion sequence 1-like DNA element causes the reversible expression of the virulence capsular antigen Vi of Citrobacter freundii in Escherichia coli.

Citrobacter freundii strain WR7004 reversibly expresses the virulence capsular antigen Vi, whose production is controlled by two distinct chromosomal loci, viaA and viaB. The rate of oscillation between the Vi-producing (Vi+) state and the Vi-nonproducing (Vi-) state in strain WR7004 ranges from 2 X 10(-4) to 7 X 10(-3) transitions per bacterium per generation. A similarly high conversion rate from Vi+ to Vi- occurs in Escherichia coli HB101 harboring pWR127, a plasmid that contains the 18-kilobase-pair (kb) viaB region cloned from WR7004. However, the Vi- state in HB101 harboring pWR127 was so stable that transition to the Vi+ state was not detected (less than 10(-10) per bacterium per generation). When pWR127 DNA derived from Vi- strains of HB101 harboring pWR127 was transformed in HB101 recipient, a small number of Vi+ transformants were seen among the transformants, which were predominantly Vi-. The viaB region consists of two EcoRI digestion fragments, A (8.6 kb) and B (9.4 kb). A discrete 700- to 800-base-pair DNA element was found to be inserted in the EcoRI B fragment of the viaB region of pWR127 when derived from Vi- strains. However, no such DNA element was found in the pWR127 DNA isolated from Vi+ strains. This discrete DNA element inserts into a recombinational hot spot 1.1 kb from the end of the EcoRI B fragment and behaves as an insertion sequence 1 (IS1)-like element. The insertion of this IS1-like element in the viaB region thus disrupts expression of the Vi antigen. Restoration of Vi expression results when this element is excised.

Antigens, Bacterial↗

PubMed1994

Targeted insertions of two exogenous collagen genes into both alleles of their endogenous loci in cultured human cells: the insertions are directed by relatively short fragments containing the promoters and the 5' ends of the genes.

Previous studies demonstrated that type II procollagen is synthesized by HT-1080 cells that are stably transfected with constructs of the human COL2A1 gene that contain the promoter and 5' end of either the COL2A1 gene or the human COL1A1 gene. Since the host HT-1080 cells were from a human tumor line that synthesizes type IV collagen but not type II or type I procollagen, the results suggested that the constructs were integrated near active enhancers or promoters. Here, however, we demonstrate that a 33-kb construct of the COL2A1 gene containing a 5' fragment from the same gene was inserted into both alleles of the endogenous COL2A1 gene on chromosome 12, apparently by homologous recombination by a nonconservative pathway. In contrast, a similar construct of the COL2A1 gene in which the 5' end was replaced with a 1.9-kb fragment from the 5' end of the COL1A1 gene was inserted into both alleles of the locus for the COL1A1 gene on chromosome 17. Therefore, targeted insertion of the gene construct was not directed by the degree of sequence homology. Instead, it was directed by the relatively short 5' fragment from the COL1A1 gene that contained the promoter and the initially transcribed sequences of the gene. After insertion, both gene constructs were expressed from previously inactive loci.

3T3 Cells↗

PubMed1999

Membrane insertion kinetics of a protein domain in vivo. The bacterioopsin n terminus inserts co-translationally.

The pathway by which segments of a polytopic membrane protein are inserted into the membrane has not been resolved in vivo. We have developed an in vivo kinetic assay to examine the insertion pathway of the polytopic protein bacterioopsin, the apoprotein of Halobacterium salinarum bacteriorhodopsin. Strains were constructed that express the bacteriorhodopsin mutants I4C:H(6) and T5C:H(6), which carry a unique Cys in the N-terminal extracellular domain and a polyhistidine tag at the C terminus. Translocation of the N-terminal domain was detected using a membrane-impermeant gel shift reagent to derivatize the Cys residue of nascent radiolabeled molecules. Derivatization was assessed by gel electrophoresis of the fully elongated radiolabeled population. The time required to translocate and fully derivatize the Cys residues of I4C:H(6) and T5C:H(6) is 46 +/- 9 and 61 +/- 6 s, respectively. This is significantly shorter than the elongation times of the proteins, which are 114 +/- 26 and 169 +/- 16 s, respectively. These results establish that translocation of the bacterioopsin N terminus and insertion of the first transmembrane segment occur co-translationally and confirm the use of the assay to monitor the kinetics of polytopic membrane protein insertion in vivo.

Apoproteins↗

PubMed2003

Insertion of tantalum beads in RSA of the hip: variations in incidence of extra-osseous beads with insertion site.

Radiostereometric analysis (RSA) is a technique that can be used to measure the in-vivo micro-motion of the components of hip arthroplasty. It requires the insertion of tantalum beads into the bone permitting analysis of the radiographs. Extra-osseous beads reduce the usable bead pattern and previous studies have reported their incidence in the proximal femur as between 11% and 13% of all inserted beads. We reviewed the relative incidence of extra-osseous beads by examining 1038 radiographs of 97 patients who were part of an ongoing RSA hip study. Extra-osseous beads were seen in 44% of our patients with most having a single extra-osseous bead. The relative incidence of extra-osseous beads was 2% and 6% in the femur and pelvis, respectively. We observed a reduction in the incidence of the femoral beads with time, but not of the pelvis, which suggests that experience in the insertion technique improves the insertion rates at least of the femur. Our findings indicate that the incidence of extra-osseous beads is lower than previously reported. However, care should be taken in the design of studies to ensure that an adequate number of beads are placed in locations which are both surgically achievable and technically useful.

Arthroplasty, Replacement, Hip↗

PubMed1981

Insertion of influenza M protein into the viral lipid bilayer and localization of site of insertion.

Recent studies with isolated M protein from influenza virus have shown that the protein has a high affinity for lipid. The ability of M to partition into lipid vesicles merely by shaking vesicles and M together is suggestive evidence that the protein could be interacting with the lipid in the virus particle. A more direct analysis was carried our here to determine whether M is in contact with the viral lipid in situ, by using the photoactivatable hydrophobic probe, pyrenesulfonyl azide. Covalent linkage of this probe to M indicated that a segment of M residues with in the virus membrane in contact with the lipid bilayer. M inserted into lipid vesicles at two locations on the molecule. A major insertion into lipid occurred in the middle of the molecule where a large cluster of 20 hydrophobic and neutral amino acids occurs. A second insertion occurred approximately one fourth in from the amino terminus, where a smaller segment of 13 uncharged amino acids is found. Confirmation that M inserted into lipid at these locations came also from results with cyanogen bromide fragments of M. Of the 12 to 13 fragments produced, 3 specifically bound to lipid vesicles. These were the first, second, and third contiguous segments beginnings at the amino terminus and containing the two hydrophobic areas noted above.

Amino Acid Sequence↗

PubMed2001

Insight into mammalian selenocysteine insertion: domain structure and ribosome binding properties of Sec insertion sequence binding protein 2.

The cotranslational incorporation of the unusual amino acid selenocysteine (Sec) into both prokaryotic and eukaryotic proteins requires the recoding of a UGA stop codon as one specific for Sec. The recognition of UGA as Sec in mammalian selenoproteins requires a Sec insertion sequence (SECIS) element in the 3' untranslated region as well as the SECIS binding protein SBP2. Here we report a detailed analysis of SBP2 structure and function using truncation and site-directed mutagenesis. We have localized the RNA binding domain to a conserved region shared with several ribosomal proteins and eukaryotic translation termination release factor 1. We also identified a separate and novel functional domain N-terminal to the RNA binding domain which was required for Sec insertion but not for SECIS binding. Conversely, we showed that the RNA binding domain was necessary but not sufficient for Sec insertion and that the conserved glycine residue within this domain was required for SECIS binding. Using glycerol gradient sedimentation, we found that SBP2 was stably associated with the ribosomal fraction of cell lysates and that this interaction was not dependent on its SECIS binding activity. This interaction also occurred with purified components in vitro, and we present data which suggest that the SBP2-ribosome interaction occurs via 28S rRNA. SBP2 may, therefore, have a distinct function in selecting the ribosomes to be used for Sec insertion.

3' Untranslated Regions↗

PubMed2002

The peripherally inserted central catheter (PICC): a prospective study of its natural history after cubital fossa insertion.

A prospective cohort study was undertaken to describe the natural history of the cubital fossa peripherally inserted central catheter (PICC), determine which factors influenced the hazard of complication and develop a standard methodology for evaluation of a PICC service. A total of 4349 patient days of PICC observation were analysed using survival analysis techniques. The median time to PICC removal for a complication was 60 days. The most common complications were phlebitis, malposition and tip migration. Complications usually occurred during the first week. There was only one episode of line-related sepsis. Size 3 French gauge catheters had a complication rate of 7.3 per 1,000 line days compared to 14.2 for 4 French catheters (hazard rate 1.26 90% CI 1.02 to 1.55). PICCs requiring two or more attempts at insertion were more likely to develop complications than those inserted at the first attempt: 20 per 1,000 line days vs 10.5 but the confidence intervals were wide (hazard rate 1.91, 90% CI 0.90 to 4.05). Operator (amongst the four experienced operators who inserted all PICCs), arm of placement, or medial or lateral placement in the cubitalfossa did not influence PICC survivaL

Adult↗

PubMedDate not supplied

Direction of catheter insertion and incidence of paresthesias and failure rate in continuous epidural anesthesia: a comparison of cephalad and caudad catheter insertion.

BACKGROUND AND OBJECTIVES: Paresthesias and unblocked segments during continuous epidural anesthesia--sometimes leading to higher doses of local anesthetics--can increase the risk of this technique. A cephalad insertion of the epidural catheter might decrease the incidence of these problems, but this has not been evaluated before in a controlled study. METHODS: In a prospective, double-blind, randomized study, we compared the incidence of paresthesias during catheter insertion and the failure rate of continuous epidural anesthesia in two groups of obstetric patients. In group 1 (n = 52), the Tuohy needle bevel was directed cephalad during catheter insertion. In group 2 (n = 53), it was directed caudad. The catheter was introduced 4-5 cm into the epidural space, and bupivacaine 25 mg plus fentanyl 50 micrograms were administered through it. RESULTS: Twenty percent of the patients in group 1 had paresthesias versus 40% in group 2 (p = 0.0249; 95% confidence interval of the difference 1-40%); intensity of paresthesias was greater in group 2. Pain relief was complete in 75% and 80% of the patients in groups 1 and 2, respectively (NS); three patients in group 1 and one in group 2 had unblocked segments (NS). CONCLUSIONS: Our results support the contention that the catheter should be directed cephalad during insertion for continuous epidural anesthesia.

Adult↗

PubMedDate not supplied

The peripherally inserted central catheter: a retrospective look at three years of insertions.

The advent of intravenous therapies administered in the home setting has created a demand for the peripherally inserted central catheter (PICC). A variety of PICCs is now on the market, offering choices of insertion methods as well as size and length. This article represents a retrospective look at 858 PICCs inserted over a 3-year period, using three insertion methods: 1) the Seldinger technique, 2) the peel-away sheath, and 3) the break-away needle. A brief history of PICCs along with problems encountered, an account of PICCs in immune-suppressed patients, and principles of catheter management are presented.

Adolescent↗

PubMed1996

Effect of bulk insertion, prepolymerized resin composite balls, and beta-quartz inserts on microleakage of Class V resin composite restorations.

Polymerization shrinkage of resin composite at the margin can seriously decrease the longevity of the restoration. Three direct restorative techniques were evaluated for their effects on microleakage. Sixty Class V cavities were prepared in extracted human maxillary molar teeth. The cavities were restored with resin composite placed in one bulk increment, resin composite and a beta-quartz insert, or resin composite and a prepolymerized resin composite ball insert. After they were thermocycled and placed in 0.5% basic fuchsin for 24 hours, the specimens were sectioned and examined for microleakage. The restorations with a prepolymerized ball of resin composite exhibited significantly more microleakage than did restorations with a beta-quartz insert or restorations inserted in bulk.

Ceramics↗

PubMed2002

Analysis of membrane topology of the human reduced folate carrier protein by hemagglutinin epitope insertion and scanning glycosylation insertion mutagenesis.

The human reduced folate carrier (RFC) is the major membrane transport system for both reduced folates and chemotherapeutic antifolate drugs, such as methotrexate (MTX). Although the RFC protein has been subjected to intensive study in order to identify critical structural and functional determinants of transport, it is impossible to assess the significance of these studies without characterizing the essential domain structure and membrane topology. The primary amino acid sequence from the cloned cDNAs predicts that the human RFC protein has 12 transmembrane domains (TMDs) with a large cytosolic loop between TMDs 6 and 7, and cytosolic-facing N- and C-termini. To establish the RFC membrane topology, a hemagglutinin (HA) epitope was inserted into the individual predicted intracellular and extracellular loops. HA insertions into putative TMD interconnecting loops 3/4, 6/7, 7/8, and 8/9, and the N- and C-termini all preserved MTX transport activity upon expression in transport-impaired K562 cells. Immunofluorescence detection with HA-specific antibody under both permeabilized and non-permeabilized conditions confirmed extracellular orientations for loops 3/4 and 7/8, and cytosolic orientations for loops 6/7 and 8/9, and the N- and C-termini. Insertion of a consensus N-glycosylation site [NX(S/T)] into putative loops 5/6, 8/9, and 9/10 of deglycosylated RFC-Gln(58) had minimal effects on MTX transport. Analysis of glycosylation status on Western blots suggested an extracellular orientation for loop 5/6, and intracellular orientations for loops 8/9 and 9/10. Our findings strongly support the predicted topology model for TMDs 1-8 and the C-terminus of human RFC. However, our results raise the possibility of an alternative membrane topology for TMDs 9-12.

Binding Sites↗

PubMed2001

NKBOR, a mini-Tn10-based transposon for random insertion in the chromosome of Gram-negative bacteria and the rapid recovery of sequences flanking the insertion sites in Escherichia coli.

We have constructed an R6K-based suicide vector that permits the random insertion of a mini-transposon named NKBOR into the chromosome of Gram-negative bacteria and the subsequent rapid cloning of sequences flanking the insertion site in Escherichia coli. This mini-transposon contains a conditional R6K plasmid origin of replication, a kanamycin resistance gene and unique restriction sites between the IS10 inverted repeats. NKBOR can be propagated by replication in an E. coli strain containing the R6K replicase pi protein. Alternatively the mini-transposon can be replicated in a pSC 101 derivative that is thermosensitive for its replication so that the mini-transposon acts as a suicide plasmid at nonpermissive temperatures. Efficient NKBOR transposition is ensured by expression of an adjacent transposase gene and has been demonstrated in E. coli, Klebsiella pneumoniae, and Erwinia carotovora. Sequences flanking the insertion sites in these strains can be rapidly recovered and identified in E. coli strains expressing the R6K pi protein.

Chromosomes, Bacterial↗