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PubMedDate not supplied

[Para-median or midline approach in the insertion of a Tenckhoff catheter in patients with ambulatory continuous peritoneal dialysis. Comparative study].

HYPOTHESIS: Insertion of a Tenckhoff catheter through a small para-median incision results in less catheter dysfunction than in cases where insertion is made through a midline incision. MATERIAL AND METHODS: This is a prospective, longitudinal, comparative, observational study of cause and effect; in other words, it is a study of two cohorts. The study included patients with chronic renal insufficiency aged 16 years and over of either sex who required insertion of a Tenckhoff catheter for peritoneal dialysis treatment. A total of 44 patients were recruited and were divided randomly into two groups: Group A, numbering 23 patients, had their catheter inserted through midline below umbilicus, while Group B numbering 21 patients had their catheter inserted through via para-median approach. The study employed Student t parametric test and chi square trial hypothesis for non-parametric variables. Rejection criterion was p < 0.05. RESULTS: In both groups, cause of chronic renal insufficiency was diabetic nephropathy. After a 30-day follow-up period, the group of patients with catheter inserted through midline incision, i.e., Group A, presented dysfunction in 43.5% of cases. In the group with catheter inserted through para-median incision, i.e., Group B, dysfunction presented in 38% of cases with no other significant statistical difference occurring. Main cause of dysfunction occurring in Group A was catheter migration, whereas in Group B the main cause was dialysis fluid leakage. CONCLUSIONS: This study concluded that there was no significant difference between the two surgical techniques employed for Tenckhoff catheter insertion with regard to incidence of catheter migration, leakage of dialysis solution, catheter obstruction due to adhesions, or post-incisional hernias during the immediate post-surgical period (30 days). Furthermore, no significant difference was found between the two groups with respect to other types of complications such as peritonitis, infection along the subcutaneous catheter pathway, or catheter obstruction due to blood clots.

Adult↗
PubMedDate not supplied

Prolonged delivery of ciprofloxacin hydrochloride from hydrophilic ocular inserts.

Ocular inserts were developed with prolonged release of drug and minimum swelling within cul-de-sac using ciprofloxacin (CPF) hydrochloride as a model drug. The ocular inserts were fahricated with sodium alginate films loaded with drug and then treated with calcium chloride. A 4% w/v solution of calcium chloride and an exposure of 15 s to this solution was found to be the optimum treatment combination of inserts. Four types of inserts were produced: type-I contained CPF hydrochloride and alginate, type-II contained CPF crystals and alginate, type-III contained CPF hydrochloride inalginate and hydroxypropylmethylcellulose (HPMC) matrix and type-IV contained CPF crystals entrapped inalginate and HPMC matrix. In vitro release profile of drug from the inserts followed Higuchi and first-order kinetic models. Longer duration for 90% drug release were obtained from types-II and IV inserts than from types I and III, although type III had a longer duration than type-I inserts. In vivo studies were carried out in rabbit eyes by measuring the tear concentrations against time. From the pharmacokinctic parameters obtained types II and IV were found to prolong the duration of action more than 2 days while types I and II inserts the duration of action lasted for about 1.5 days.

Administration, Topical↗
PubMed2006

[Visual functions' detailed evaluating in patients with Sjögren's syndrome before and after intracanalicular implants' (Smart Plug) insertion--(first results)].

UNLABELLED: The aim of the study was to determine exact visual functions (log MAR [minimal angle of resolution] and CS [contrast sensitivity]) and to evaluate corneal topographic maps in patients with established (by means of laboratory and biopsy examinations) Sjogren's Syndrome, and to determine the difference in subjective symptoms before and after insertion of the intracanalicular implants as well. PATIENTS AND METHODS: Twelve eyes (1 man, 6 women) with established Sjogren's syndrome were examined before and during two months after the insertion of the plugs. The best-corrected visual acuity (BCVA) was assessed on Landolt C rings optotypes. CS was measured on computer-controlled device (Neuroscientific Corp., U.S.A.) in 6 space-frequencies (0.74-29.55 c/deg). The corneal topographic changes (Keraton Opticon) were established by means of comparing total aberrations values before and after the intracanalicular implants' (Smart Plugs type) insertion. The control group for visual functions assessment consisted of 10 woman (20 eyes) of similar middle age. RESULTS: The BCVA on log MAR optotypes was 0.84 (0.69-0.95) before and 0.88 (0.52-1.23) after the insertion, on both occasions, it was lower than in the control group. The CS was before the insertion in all of the spatial frequencies lower, the largest differences were in the frequencies range 1.97-7.29 c/deg (p < 0.01). After the treatment, the values grow but they don't reach the values of the control group. Subjective complains of the patients decreased markedly; in 100% they had relief, in 3 cases they referred improvement up to 50%; no patient observed tearing. The frequency of drops' application has decreased by 63%. The Schirmer test, in 100% positive before the treatment, was after the insertion in 75% negative; the height of the tear-meniscus was positive in 100% before the procedure, and after that, its measurement improved to 1 mm in 91%; in 9% it was 1.5 mm. We also noticed changes of the ocular surface by means of lissamine green staining; this test was before the procedure positive in 100%, the improvement after that was in 63%. The regularity of the corneal surface is the determining factor of visual functions in "dry eyes". The measurement of the corneal topography is useful in differential diagnosis and helps to distinguish mild and more serious conditions of dry eye. The improvement of BCVA and CS values after insertion corresponds with patients' subjective evaluating. The best value of the treatment is improvement of the patients' comfort; furthermore, after insertion of the permanent plugs they feel pronounced subjective relief and lowering of the frequency of drops application.

Adult↗
PubMed2006

[Random comparative study between intrauterine device Multiload Cu375 and TCu 380a inserted in the postpartum period].

OBJECTIVE: To evaluate safety and effectiveness of the intrauterine device Multiload Cu375 compared with the TCu 380A inserted in the postpartum period. PATIENTS AND METHODS: In a randomized comparative study carried out in the National Perinatology Institute, intrauterine devices MLCu 375 and Tcu 380A were inserted to 157 patients who voluntary accepted, and previously signed informed consent. There were four instances for the intrauterine devices insertion: within 10 minutes after vaginal delivery, during cesarean section (immediate postplacental insertion) and postpartum-postcesarean insertion (in the time range of 10 min to 48 h). All insertions were made with ring forceps. From 1 h to 24 h later, abdominal ultrasound examinations were performed to assess the distances between the upper part of the device to the fundus of uterine cavity. Follow up visits were scheduled at 3, 6, 9 and 12 months. Net cumulative life table event rates of discontinuations were estimated at one year. RESULTS: The expulsion rates were 10.4 for the MLCu 375 and 7.7 for the TCu 380A and they were not influenced by the moment of the intrauterine device insertion, not by the cervical dilatation, neither by the distance of the intrauterine device to the fundus of uterine cavity. The removal rates for bleeding and pain were 4.9 and 4.8, the removal rates for non medical reasons were 3.7 and 4.9 respectively. There was one case of genital infection in the MLCu 375 group. There were no pregnancies, nor uterine perforation. The one year continuation rates were 77.1 and 82.6 respectively. There were no statistical significant differences in the comparative rates. CONCLUSIONS: The intrauterine device MLCu 375 is as safe and effective as the TCu380A when they are inserted in the postpartum period.

Adult↗
PubMed1991

[Insertion anomalies of the horizontal muscles and dysfunctions of the oblique muscles in the A-V patterns].

Insertion anomalies of the horizontal rectus muscles and dysfunctions of the oblique muscles were studied in 141 cases with A-V patterns of 824 patients with horizontal strabisums. The frequency of insertion anomaly among all cases of A-V pattern studied was 49.6%, there were more V pattern insertion anomalies than in those of A pattern, but the ratio of insertion anomalies in each type of A-V pattern was more in the A pattern than in the V pattern. In the oblique muscles, overaction of the inferior oblique muscle in the V pattern and of the superior oblique muscle in the A pattern were frequently encountered and insufficient action of the oblique muscles occurred more frequently in cases A-V esotropia than in cases of exotropia. In cases of combination of insertion anomaly of the horizontal rectus muscles with dysfunction of the oblique muscles, insertion anomaly was involved in 91.3% of cases of overaction of the superior oblique muscles, but only in 37.4% of cases of overaction of the inferior oblique muscles. The combination of insertion anomalies with dysfunction of the oblique muscles suggests a possible existence of simulated dysfunction of the oblique muscles in some cases of dysfunction of the oblique muscles diagnosed preoperatively. At the choice of a procedure in surgical correction, a confirmation of insertions of the horizontal muscles during operations is needed, and for this purpose the perilimbal incision is more appropriate than the fornix incision.

Eye Movements↗
PubMed1990

Generation of a 50,000-member human DNA library with an average DNA insert size of 75-100 kbp in a bacteriophage P1 cloning vector.

A bacteriophage P1 cloning system that permits the isolation and amplification of cloned DNA fragments as large as 100 kbp was described previously. We have now utilized a similar system to generate a 50,000-member human DNA library with DNA inserts ranging in size from 75 to 100 kbp. Two major obstacles were overcome in constructing the library. The first concerned the mcrAB restriction system of Escherichia coli, which degrades DNA containing MeC and interferes with the recovery of cloned human DNA inserts. In the P1 cloning system, the effect of the Mcr restriction activity is to decrease recovery of cloned inserts by about 35-fold when the activity is in the host cell line and by about 3-fold when the activity is in the cells used to prepare the packaging extract. To circumvent this problem we inactivated, by mutation, the McrAB proteins in both components of the cloning system. The second obstacle concerned the preferential cloning of small DNA fragments from a population of fragments ranging in size from 20 to 100 kbp. To deal with this problem we first modified the P1 lysogen used to prepare the in vitro head-tail packaging extract so that it would produce 12 times as many large P1 heads (head capacity about 110 kbp) as small P1 heads (head capacity about 45 kbp). We then restructured the P1 cloning vector so that it could be used to produce vector "arm" fragments that could be ligated to insert DNA at only one end. This prevented the formation of long concatamers consisting of alternating units of vector and insert DNA and prohibited the packaging of small inserts in large phage heads. Using the insert-biased large head extract, the arms vector, and size-selected human DNA fragments, we showed that as much as 90% of recovered transformants contained inserts in the desired high molecular weight range.

Cloning, Molecular↗
PubMed1979

Further characterization of a depurinated DNA-purine base insertion activity from cultured human fibroblasts.

The purification from cultured human fibroblasts of a protein that binds specifically to partially depurinated DNA and inserts purines into those sites is described. The purine insertion, but not the binding, requires K+. The DNA binding can be saturated with increasing apurinic sites and is weakened by the presence of adenine or guanine. Base insertion into depurinated DNA is specific for adenine or guanine; none is observed with dATP or dGTP. When the depurinated DNA substrate is specifically cleaved with apurinic endonuclease, no purine insertion occurs. Guanine insertion does not occur into tRNA or depyrimidinated DNA, and thymine is not inserted into either depyrimidinated DNA or depurinated DNA. Purine insertion activity follows Michaelis-Menten kinetics with respect to purintes; the apparent Km values for both adenine and guanine are 5 microM. The enzyme binds the purine bases very tightly. Adenine binding saturates at less than 1 microM adenine, perhaps reflecting the low intracellular adenine concentration. The binding protein specific for UV-irradiated DNA (Feldberg, R.S., and Grossman, L. (1976) Biochemistry 15, 2402-2408) had no detectable purine or pyrimidine base insertion activity with depurinated or depyrimidinated DNAs.

Apurinic Acid↗
PubMed1988

168 double J (pigtail) ureteric catheter insertions: a retrospective review.

The medical records of 116 patients who had 168 ureteric double J (pigtail) catheters inserted over a 6-year period between 1981 and 1987 were reviewed. Eighty-five patients had pigtails inserted for benign conditions and 31 for malignant disease. The pigtail catheters were inserted cystoscopically in 88 patients, percutaneously in 7 patients and by open surgery in 21 patients. Of 168 pigtail catheters used, 147 were soft (silicone, multilength) and 21 hard (polyurethane). No mortality was attributable to the use of these catheters but certain complications were commonly encountered. Loin discomfort occurred after 32 (19%) insertions; 27 of 147 (18%) soft catheters and 5 of 21 (24%) hard catheters. Trigonal irritation, confirmed cystoscopically was reported in 26 of 147 (18%) insertions of soft catheters and in 6 of the 21 (29%) with hard catheters (chi 2, P = 0.37). Urinary tract infection (confirmed by urine microscopy and culture) occurred after 46 (31%) soft catheter insertions and after 13 of 21 (61%) hard catheter insertions (chi 2 test, P = 0.01). Stent migration occurred in five patients and obstruction in two. Pigtail catheters are safe ureteric stents which are easy to insert and their use is supported by this study. The complications of associated infection, trigonal irritation and loin discomfort are relatively common and still occur even with soft catheters. Careful monitoring of all patients with pigtail catheters in position is recommended.

Adult↗
PubMed1987

Nucleotide insertion kinetics opposite abasic lesions in DNA.

A gel assay is introduced to measure DNA polymerase insertion kinetics at single sites along a DNA template strand. The assay is used to analyze the kinetics of inserting deoxynucleotides opposite a synthetic abasic (apurinic/apyrimidinic) lesions using Drosophila DNA polymerase alpha. The location of the abasic lesion next to different nearest-neighbor bases allows the effects of base stacking on the specificity of insertion to be evaluated. The specificity of nucleotide insertion, Vmax/Km, is 6-11 times greater for A over G and about 20-50 times greater for A over C and T. The insertion specificity at the abasic lesion appears to depend more on differences in Vmax than Km. Apparent Michaelis constants for inserting A and G deoxynucleotides are similar to within about a factor of 2. The insertion of A or G occurs most efficiently at the abasic lesion when T is the 5'-nearest neighbor on the primer strand and least efficiently when G is the 5'-nearest neighbor. The presence of different base stacking partners adjacent to the site of insertion has up to a 4-fold effect on specificity.

Animals↗
PubMed1982

Association between microorganism growth at the catheter insertion site and colonization of the catheter in patients receiving total parenteral nutrition.

Catheter-related sepsis is one of the major complications of total parenteral nutrition (TPN) therapy. The relationship between microbial colonization of the skin at the site of catheter insertion and colonization of the central venous catheter was investigated in 74 catheters used to administer TPN therapy in 53 patients. Semiquantitative culture specimens were obtained from the insertion site and intravascular and subcutaneous catheter segments at the time of catheter removal. Bacteria and/or fungi were recovered from 19 catheters and 19 insertion sites; of the 19 colonized catheters, 6 had sterile insertion sites. Organisms isolated from the remaining 13 catheters were isolated concurrently from the insertion site. Catheter-associated bacteremia or fungemia was observed in 10 of the 19 patients with colonized catheters. The association between colonization of catheters and the presence of more than 10(3) bacterial or fungal colony-forming units at the insertion site was significant (P less than 0.005). These results demonstrated that colonization of catheters by organisms present on the skin at the site of catheter insertion occurred twice as frequently as colonization by the hematogenous route. The results also suggested that colonization of catheters by organisms present at the insertion site occurred only after a threshold number of organisms was reached.

Adult↗
PubMed1982

Synthesis and maturation of the erythrocyte anion transport protein--an internal sequence for membrane insertion.

The biosynthesis of the erythrocyte anion transport glycoprotein, Band III (Mr 100,000), is of interest, as its N-terminal half is hydrophilic and faces the cytoplasmic surface; the C-terminal half spans the phospholipid bilayer several times. Band III is synthesized by erythroid precursor cells obtained from the spleens of anaemic mice. Newly synthesized Band III was inserted into rough endoplasmic reticulum membranes with an asymmetric orientation which resembled that of mature Band III in erythrocyte membranes: the N-terminal portion of the molecule facing the cytoplasm. Newly made Band III contained a high-mannose asparagine-linked oligosaccharide, which was susceptible to cleavage by endoglycosidase H. During the next 20-30 min, this oligosaccharide was processed to a form resistant to endoglycosidase H degradation, presumably in the Golgi complex. The processed Band III was subsequently expressed on the cell surface, at about 30-45 min after synthesis. To study the mechanism of insertion of Band III into microsomes, we used erythroid precursor cells from the spleens of anaemic mice as a source of messenger RNA for studies in vitro in the wheat germ and reticulocyte lysate cell-free system containing dog pancreatic microsomes. Immediately after synthesis, Band III was found to be inserted into microsomal membranes in its mature configuration, with the N-terminal portion exposed to the cytoplasm and its hydrophobic C-terminal portion spanning the lipid bilayer. The newly-synthesized Band III was also provided with a high-mannose asparagine-linked oligosaccharide. Band III was found to be inserted into dog pancreatic microsomes in a co-translational manner; in synchronized translation studies microsomes could be added as late as the time when the hydrophilic N-terminal half of the protein had been synthesized and still allow normal trans-membrane insertion and glycosylation. There is no cleavage of any N-terminal peptide during membrane insertion. In many respects, therefore, the biosynthesis of Band III resembles that of co-translationally-inserted proteins whose N-terminal portions are exposed on the exterior of the cell, like vesicular stomatitis virus glycoprotein, HLA-A antigens, and glycophorin. However, our results suggest that Band III contains a sequence near the middle of the protein which directs its insertion into endoplasmic reticulum membranes.

Acetylglucosaminidase↗
PubMed1995

Biomechanical analysis of bone mineral density, insertion technique, screw torque, and holding strength of anterior cervical plate screws.

The bone mineral density (BMD) of 99 cadaveric cervical vertebral bodies (C3-7) was determined using dual x-ray absorptiometry. The vertebral bodies were randomly assigned to receive either a unicortical (51 bodies) or bicortical (48 bodies) Caspar cervical plating screw. The initial insertion torque was measured using a digital electronic torque wrench, and the force required to withdraw the screw from the vertebral body was determined. The mean BMD for the total group of 99 was 0.787 +/- 0.154 g/cm2, the mean insertion torque was 0.367 +/- 0.243 newton-meters, and the mean pullout force was 210.4 +/- 158.1 newtons. A significant correlation was noted between BMD and torque (p < 0.0001, r = 0.42), BMD and pullout force (p < 0.0001, r = 0.54), and torque and pullout force (p < 0.0001, r = 0.88). Although the BMD of the unicortical and biocortical groups was equivalent (p = 0.92), the insertion torque and pullout force differed significantly (p = 0.02 and p = 0.008, respectively) for the unicortical and bicortical groups. A holding index for each screw and insertion technique was defined as the product of the BMD and insertion torque. The calculated holding index and resultant pullout force were significantly correlated for both techniques of screw insertion (r = 0.92), and a significant difference in holding index was observed with unicortical versus bicortical screw placement (p = 0.04). The determination of BMD and measurement of insertion torque to create a unique holding index provides an assessment of bone-screw interaction and holding strength of the screw, both of which impact on the resultant stability of cervical instrumentation. As the number of cervical plating systems increases, the determination of a holding index for various screws and insertion techniques may assist in the comparison of cervical instrumentation.

Absorptiometry, Photon↗
PubMed1996

Quantitative comparison of soft tissue-bone interface at chondral ligament insertions in the rabbit knee joint.

At chondral ligament insertions the calcified fibrocartilage interdigitates deeply with the lamellar bone. The shape of this interface is formed under physiological loading conditions. For the purpose of morphological comparison between different ligament entheses in the rabbit knee, the number and frequency of interdigitations and thickness of calcified fibrocartilage were quantitated at the femoral insertion of the medial collateral ligament, both insertions of the cruciate ligaments, and the tibial insertion of the patellar ligament. Among the insertions, the femoral insertion of the medial collateral ligament showed the lowest frequency and depth of interdigitations at the soft tissue-bone interface, but had the thickest zone of calcified fibrocartilage. An inverse relationship was found at the insertion interface of the cruciate and patellar ligaments. The frequency and depth of interdigations at the bone-soft tissue interface at different chondral entheses seem to be related to the mechanical strength of the respective ligament; meanwhile it may be hypothesised that the thickness of the calcified fibrocartilage might be more related to the amount of motion which takes place at an insertion.

Animals↗
PubMed1997

Tibial insert undersurface as a contributing source of polyethylene wear debris.

Sixty-seven ultrahigh molecular weight polyethylene tibial inserts from cementless total knee arthroplasties were retrieved at autopsy and revision surgery and analyzed for evidence of articular and nonarticular surface wear after a mean implantation time of 62.8 months (range, 4-131 months). Polyethylene cold flow and abrasive wear on the nonarticular insert surface (undersurface) were assigned a wear severity score (Grade 0-4). The severity of articular wear was assessed quantitatively and graded. Corresponding prerevision radiographs were evaluated for evidence of tibial metaphyseal osteolysis and osteolysis around tibial fixation screws. Exact nonparametric conditional inference methods were used to establish correlations between different variables and the occurrence of tibial metaphyseal osteolysis. Severe Grade 4 wear of the tibial insert undersurface was associated with tibial metaphyseal osteolysis or osteolysis around fixation screws. Time in situ statistically was related to Grade 4 undersurface wear and tibial metaphyseal osteolysis. The occurrence of tibial osteolysis was not related statistically to articular wear severity, insert thickness, or implant type. The main articulation between the femoral implant and ultrahigh molecular weight polyethylene insert has been assumed to be the primary source of polyethylene debris contributing to osteolysis and total knee arthroplasty implant failure. The undersurface of the insert is an additional source of polyethylene debris contributing to tibial metaphyseal osteolysis. To lessen polyethylene debris produced at this modular interface, the tibial implant locking mechanism should fix the insert firmly to the metal backing to decrease relative micromotion. Because motion between the insert and metal backing may be inevitable, the wear characteristics of the inner tray surface should be optimized to minimize wear debris production at this other articulation.

Arthroplasty, Replacement, Knee↗
PubMed2004

Transposon vectors for gene-trap insertional mutagenesis in vertebrates.

The function of most vertebrate genes remains unknown or uncertain. Insertional mutagenesis offers one approach to identify and understand the function of these genes. Transposons have been used successfully in lower organisms and plants for insertional mutagenesis, but until activation of the Sleeping Beauty (SB) transposon system, there was no indication of active DNA-based transposons in vertebrates. Investigator-driven insertional mutagenesis in vertebrates has relied on retroviral insertions or selection of low-frequency integration of naked DNA in ES cell lines. We have combined the highly active SB transposon with gene-trapping technology to demonstrate that transposon traps can be used for insertional mutagenesis screens in vertebrates. In our studies about one-fourth of the trap insertions appear to be in transcriptional units, a rate that is commensurate with random integration. We show that gene-traps coupled to a fluorescent protein reporter gene can be used to detect insertions into genes active in specific cells of living zebrafish embryos, supporting use of our transposon traps for high-throughput functional genomic screens in vertebrates.

Animals↗
PubMed2004

Probing the alpha-complementing domain of E. coli beta-galactosidase with use of an insertional pentapeptide mutagenesis strategy based on Mu in vitro DNA transposition.

Protein structure-function relationships can be studied by using linker insertion mutagenesis, which efficiently identifies essential regions in target proteins. Bacteriophage Mu in vitro DNA transposition was used to generate an extensive library of pentapeptide insertion mutants within the alpha-complementing domain 1 of Escherichia coli beta-galactosidase, yielding mutants at 100% efficiency. Each mutant contained an accurate 15-bp insertion that translated to five additional amino acids within the protein, and the insertions were distributed essentially randomly along the target sequence. Individual mutants (alpha-donors) were analyzed for their ability to restore (by alpha-complementation) beta-galactosidase activity of the M15 deletion mutant (alpha-acceptor), and the data were correlated to the structure of the beta-galactosidase tetramer. Most of the insertions were well tolerated, including many of those disrupting secondary structural elements even within the protein's interior. Nevertheless, certain sites were sensitive to mutations, indicating both known and previously unknown regions of functional importance. Inhibitory insertions within the N-terminus and loop regions most likely influenced protein tetramerization via direct local effects on protein-protein interactions. Within the domain 1 core, the insertions probably caused either lateral shifting of the polypeptide chain toward the protein's exterior or produced more pronounced structural distortions. Six percent of the mutant proteins exhibited temperature sensitivity, in general suggesting the method's usefulness for generation of conditional phenotypes. The method should be applicable to any cloned protein-encoding gene.

Amino Acid Sequence↗
PubMedDate not supplied

DNA insertions in the 'silent' regions of the 2 microns plasmid of Saccharomyces cerevisiae influence plasmid stability.

The 2 microns plasmid of the yeast Saccharomyces cerevisiae is in principle a suitable vector for expression of foreign genes, due to its high copy number and extreme stability. However, the cloning of genes into 2 microns often results in a reduced copy number and/or reduced stability. One reason for this observed instability could be that the inserts in general were made in one of the several open reading frames (ORFs) of the plasmid. Therefore we studied the effect on stability of insertions in the silent regions of 2 microns without interrupting any known essential regions or ORFs. Using the SnaBI site, a yeast-integrating plasmid (Yip5) was introduced into the region between the ARS and STB locus in two possible orientations. The resulting plasmids could be stably maintained in the cells without the need for complementation by the wild-type 2 microns plasmid. However, the stability of these plasmids in a cir. host was still one to two orders of magnitude lower (0.2% and 0.8% respectively) as reported for the wild-type 2 microns (0.01%). Removal of 2 kb of the bacterial sequences from Yip5 did not increase stability. The stability was dependent on the orientation of the insert. We found that in the less stable orientation, transcription originating from the insert was running into the STB region. DNA inserted in the XmaIII site located outside the ORFs in the REP2/FLP intergenic region influenced both stability and copy number of the plasmid. These effects are strongly dependent on the size of the insert. Insertion of a 2 kb DNA fragment increased the copy number, probably through an effect on FLP expression.

Blotting, Northern↗
PubMed2006

Dissociation (Ds) constructs, mapped Ds launch pads and a transiently-expressed transposase system suitable for localized insertional mutagenesis in rice.

We have developed a transiently-expressed transposase (TET)-mediated Dissociation (Ds) insertional mutagenesis system for generating stable insertion lines in rice which will allow localized mutagenesis of a chromosomal region. In this system, a Ds containing T-DNA construct was used to produce Ds launch pad lines. Callus tissues, from single-copy Ds/T-DNA lines, were then transiently infected with Agrobacterium harbouring an immobile Ac (iAc) construct, also containing a green fluorescent protein gene (sgfpS65T) as the visual marker. We have regenerated stable Ds insertion lines at a frequency of 9-13% using selection for Ds excision and GFP counter selection against iAc and nearly half of them were unique insertion lines. Double transformants (iAc/Ds) were also obtained and their progeny yielded approximately 10% stable insertion lines following excision and visual marker screening with 50% redundancy. In general, more than 50% of the Ds reinsertions were within 1 cM of the launch pad. We have produced a large number of single-copy Ds/T-DNA launch pads distributed over the rice chromosomes and have further refined the Ds/T-DNA construct to enrich for "clean" single-copy T-DNA insertions. The availability of single copy "clean" Ds/T-DNA launch pads will facilitate chromosomal region-directed insertion mutagenesis. This system provides an opportunity for distribution of gene tagging tasks among collaborating laboratories on the basis of chromosomal locations.

Chromosome Mapping↗