Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “insertion”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

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

At least 307 records · Page 17Linked to original sources
PubMed1999

A comparison of inter-aural attenuation with the Etymotic ER-3A insert earphone and the Telephonics TDH-39 supra-aural earphone.

One of the many reported advantages of the insert earphone over the supra-aural earphone is increased inter-aural attenuation (IA). Minimum values of IA determine the need for masking of the non-test ear in air-conduction audiometry. The aim of the present study was to measure inter-aural attenuation for the Etymotic Research ER-3A insert earphone (with deep and shallow insertion of the ear plug within the ear canal) and compare this with the supra-aural Telephonics TDH-39/MX41-AR earphone/cushion combination. Subjects were 18 adults ranging in age from 38 to 68 years (mean 50 years). Each subject had no hearing in one ear following translabyrinthine surgery for removal of an acoustic neuroma. The opposite ear had hearing thresholds better than 40 dB HL and an air-bone gap of less than 10 dB at any audiometric frequency. Pure tone air-conduction thresholds were obtained in the range 0.25-8 kHz. Deep insertion of the insert earphone was deemed to occur when the outside edge of the ear plug was flush with the entrance of the ear canal. Shallow insertion was deemed to occur when half of the ear plug (6 mm) was inside the entrance of the ear canal. IA was defined operationally as the difference between the good-ear and poor-ear not-masked air conduction threshold for a given audiometric frequency and earphone. The results show that the TDH-39/MX41-AR combination provides a median IA of approximately 60 dB with a lower limit of approximately 45 dB. Greater IA was obtained with the ER-3A insert earphone but this depended on the depth of insertion. With a deep insertion, the 1A values were some 15-20 dB greater than with the supra-aural earphone. Although frequency-specific IA values are provided, a simple rule of thumb is to apply masking to the non-test ear when the pure tone airconduction signal from the ER-3A insert earphone exceeds the bone conduction threshold of the non-test ear by 55 dB HL or more. If it is not possible to obtain a deep insertion depth this value should be reduced by 5 dB.

Acoustic Stimulation↗
PubMed2000

Experience with IUCD insertion outside of menses in Kenya.

OBJECTIVE: To determine if women receiving intrauterine devices (IUCDs) outside of menses have an acceptable rate of insertion problems and subsequent IUCD-related complications. DESIGN: Cross-sectional and prospective cohort study of insertions at times other than during menses. SETTING: The study was carried out in two government family planning (FP) clinics in Nairobi, Kenya. SUBJECTS: After appropriate pre-test and post-test HIV counselling, 1686 women requesting IUCDs at two FP clinics between 1994 and 1995 in Nairobi were enrolled at baseline into a study examining the effect of human immuno-deficiency virus (HIV) infection on IUCD-related complications. Six hundred and forty nine women (156 HIV-infected and 493 HIV-uninfected) were selected for the four month follow up study. They were classified according to their menstrual cycle status at time of IUCD insertion. MAIN OUTCOME MEASURES: Problems at the time of insertion (pain, bleeding, immediate expulsion) and IUCD-related complications through four months. RESULTS: Rates of immediate insertion problems were low in the women who had insertions during menses (7.0%), outside of menses (4.0%) or had oligomenorrhea/amenorrhea (2.6%). The adjusted odds ratios for IUCD insertion problems outside of menses and in oligomenorrhea/amenorrhea (versus women with insertion during menses) were 0.54 (95 % CI 0.18-1.59) and 0.39 (95% CI 0.12-1.29) respectively. IUCD-related complications were higher in the oligomenorrhea/amenorrhea (11.5%) or insertion outside of menses (6.9%), than the within menses (4.3%) groups. However, the differences were not statistically significant. Adjusted odds ratios for IUCD outside of menses and oligomenorrhoea/amenorrhea groups were 1.65 (95% CI 0.21-12.91) and 2.72 (95% CI 0.34-21.71) respectively. CONCLUSION: The results confirm that the IUCD can be safely inserted outside of menses with minimal insertion difficulties and subsequent complications. Availability of IUCDs outside of menses may enhance IUCD acceptance in Kenya and create better opportunity for visual screening of the cervix for sexually transmitted infections.

Adult↗
PubMed2001

[Insertion of intrauterine device in general practice. Which women choose the IUD, and how is the procedure done?].

INTRODUCTION: In Denmark most IUDs are inserted by general practitioners (GPs). The aim of this study was to throw light on which women choose IUD, the procedure used, and difficulties associated with insertion. MATERIAL AND METHODS: All 232 GPs in Frederiksborg County were invited to participate and 88 agreed. The GP filled in a questionnaire concerning the type of IUD, the age, parity, etc, of the woman and information about the insertion. RESULTS: Eighty-eight GPs contributed with 562 IUD insertions. IUD was mostly used by women above 25 years of age and by women who had given birth to a child. There were no differences in the insertion procedures used by the doctors. Fifty-nine per cent of the women were previous IUD users. Of the former users, 37% reported problems with the previous use of the IUD. Twenty-five per cent of the women in this study reported that they had had heavy or long-lasting menstruation before insertion. Fifty-five per cent of the women had a test for chlamydia before insertion. Twenty-three (4.1%) insertions were abandoned. 26 (4.6%) insertions were described as difficult. The highest frequency was in women using gestagen-releasing IUDs. CONCLUSION: IUD was mainly used by women, who had given birth to a child. The gestagen releasing IUD was mostly used by the elder group and by women with heavy bleedings before insertion. The procedure was very identical and the number of abandoned and difficult insertions were low and only related to the type of IUD. More female than male doctors participated. Doctors working in partnerships participated more frequently than doctors working alone.

Adult↗
PubMedDate not supplied

Mechanical performance of inflatable inserts used in limb prosthetics.

Inflatable insert products used to overcome residual limb shrinkage were evaluated in a bench test environment under compressive loading conditions. Pressure-loss tests under static loading demonstrated that after inserts were inflated to 43.4 to 45.6 kPa, insert pressures reduced from 0.09%/min to 1.36%/min in the first 5 min and from 0.00%/min to 0.27%/min in the subsequent 55 min. As inserts were inflated, they demonstrated at least two phases in their pressure versus thickness curves: A relatively low-slope portion (Phase I) was followed by a high-slope portion (Phase II). The transition from Phase I to Phase II occurred at more than a 12-mm thickness, a thickness greater than that considered acceptable for practical clinical use (10 mm). This result suggests that in a socket, stress to resist insert expansion is taken by the residual limb and socket more than by the insert itself. Cyclic-loading tests under constrained thickness conditions demonstrated that local stiffness was more sensitive to insert pressure than to constraint spacing (insert thickness). The static and dynamic test results help to explain why some users claim that inserts do not provide equal and consistent support unless inflated to a very high insert pressure. An insert that allowed adjustment of the location of the Phase I to Phase II transition point in the pressure versus thickness curve might help to overcome these limitations.

Amputation, Surgical↗
PubMed2004

[Effects of intrauterine contraceptive device on expression of vascular endothelial growth factor, kinase insert domain-containing receptor and microvessel density in endometrium].

OBJECTIVE: To investigate the expression of vascular endothelial growth factor (VEGF) and its receptor, kinase insert domain-containing receptor(KDR) and microvessel density (MVD) in endometrium from women wearing fixed copper-intrauterine contraceptive device (IUD, FCu-IUD) or fixed indomethacin-releasing copper-IUD (FICu-IUD). METHODS: Twenty healthy women were divided into two study groups: 10 cases wearing the FCu-IUD (FCu-IUD group), 10 cases wearing the FICu-IUD (FICu-IUD group). Immunohistochemical technique was used to determine the expression of VEGF and KDR in endometrium, and the microvessel density (MVD) was counted. The expression of VEGF mRNA was determined by in situ-hybridization. RESULTS: Before insertion of FCu-IUD, the expression of VEGF and KDR proteins was 0.357 +/- 0.032 and 0.215 +/- 0.029 respectively. After insertion of FCu-IUD, the expression of VEGF and KDR proteins was 0.568 +/- 0.027 and 0.244 +/- 0.022 respectively, significantly higher than before insertion (P < 0.05). The expression of VEGF mRNA was 0.359 +/- 0.022 before insertion of FCu-IUD, after insertion of FCu-IUD, the expression of VEGF mRNA was 0.425 +/- 0.019 (P < 0.05). There were no significant changes in the level of VEGF protein and mRNA, as well as KDR in endometrium before and after insertion of FICu-IUD. Compared with before insertion of FCu-IUD 15.4 +/- 2.8, a significant increase in MVD was observed after insertion of FCu-IUD 19.8 +/- 4.8, and the expression of VEGF protein was positively correlated with MVD (r = 0.847, P < 0.01). MVD counts were not different significantly before and after insertion of FICu-IUD. CONCLUSIONS: FCu-IUD can enhance the expression of VEGF and KDR in the endometrium. FICu-IUD can inhibit the activity of VEGF and KDR by releasing indomethacin. VEGF and KDR may be related to the structural and functional changes of microvessels in endometrium after insertion of FCu-IUD or FICu-IUD.

Adult↗
PubMed2004

Improving the success rate of laryngeal mask airway insertion during etomidate induction by using fentanyl or succinylcholine.

BACKGROUND: Smooth and successful insertion of a laryngeal mask airway (LMA) during etomidate induction requires a proper mouth opening and efforts to minimize airway reflexes such as gagging, coughing, or laryngospasm. We hypothesized that the concurrent use of fentanyl or succinylcholine with etomidate, the induction agent, could reduce the occurrence of airway reflexes and increase the success rate of LMA. METHODS: Sixty adult patients, ASA class I-II, scheduled for surgery under general anesthesia, were randomly allocated into three groups: group I in which patients were given etomidate 0.3 mg/kg alone; group 2 in which patients were given fentanyl 2 microg/kg and etomidate 0.3 mg/kg; groups 3 in which patients were give etomidate 0.3 mg/kg and succinylcholine 1 mg/kg. All patients were premedicated with atropine 0.01 mg/kg. When induction of anesthesia began we recorded the time from administering the designated regiminal agent or agents to the time of relaxation of jaw (the ease of mouth opening) and checked the patient for any excitatory movement after administering the induction agents. After inserting the LMA, we checked for signs of airway reflexes. Student's t test, Chi-square and Fisher's exact test were used to analyze the data. A value of P < 0.05 was considered significant. RESULTS: There were no significant differences in demographic data among the the three groups. The concurrent use of fentanyl or succinylcholine with etomidate during induction could significantly reduce the occurrence of excitatory movements and airway reflexes after LMA insertion, and increase the success rate of LMA insertion in comparison with using etomidate alone. Comparing the two adjutant agents, the concurrent use of succinylcholine could provide a significantly better jaw relaxation, shorten the time to insert the LMA and increase the success rate of LMA insertion. CONCLUSIONS: As an induction agent to facilitate insertion of LMA, etomidate alone was far from perfect. The concurrent use of 2 microg/kg of fentanyl with etomidate might significantly reduce the occurrence of airway reflexes in response to LMA insertion and increase the success rate of insertion. However, concurrent use of 1 mg/kg succinylcholine with etomidate might provide better results in terms of shortened time for the LMA insertion, jaw relaxation, and the success rate of LMA insertion than that of fentanyl.

Adult↗
PubMed1980

Migration of tendinous insertions. I. Cause and mechanism.

The cause and mechanisms of the migration of tendons and ligaments were studied in young rabbits. Three techniques were used: (1) Marking of insertions, the neighbouring periosteum and the diaphysis with metallic markers. (2) Marking of insertion sites by tetracycline as an indicator of osteogenesis. (3) Histological examination. The insertions used in the study were of three different characters: (1) Insertions subject to muscular traction (patellar ligament, quadratus femoris muscle, tibialis anterior muscle). (2) The distal insertions of the medial collateral ligament of the knee, stretched by the activity of the proximal epiphyseal cartilage of the tibia. (3) The proximal and distal insertions of the anterior annular ligament of the tibia, inserted solely in bone and periosteum. The cause of migration is the growth of periosteum dragging the insertions during its stretching, caused itself by the activity of the epiphyseal plates. The local mechanism governing migration while ensuring a continuous connexion with the bone is not the same in all sites. It depends upon the character of the bony surface at the insertion and of the function of the insertion zone, which can be osteogenic, resorptive or both. A plexus of precollagenous fibres is present at all resorptive insertion sites, and at some of the osteogenic sites.

Animals↗
PubMed1996

Effect of hearing aid experience on preferred insertion gain selection.

The effect of hearing aid experience (measured as years of hearing aid use and user insertion gain at octave frequencies from 250 Hz to 4000 Hz) on the selection of preferred insertion gain under six listening conditions and one vocalization condition was examined. Thirteen experienced hearing aid users selected their preferred insertion gain in each test condition using a modified simplex procedure. The results showed that for the listening conditions, preferred insertion gain was highly correlated with the subjects' hearing loss from 500 Hz to 2000 Hz. Hearing aid experience did not correlate at all with preferred insertion gain. On the other hand, user insertion gain at 250 Hz and 500 Hz correlated highly with preferred insertion gain selected during vocalization. Years of hearing aid use did not correlate with preferred insertion gain selection. These results suggest that user insertion gain can affect preferred insertion gain selection only when the test conditions are identical to those that the hearing aid wearers experience in everyday lives. In typical laboratory conditions, preferred insertion gain is likely determined by the stimulus characteristics and subjective preference.

Adult↗
PubMed1997

[Comparison of two techniques used in immediate postplacental insertion of TCu 380A intrauterine device: 12 month follow-up of 910 cases].

OBJECTIVE: To compare the efficacy of hand-insertion and ring forceps-insertion of TCu 380A intrauterine device (IUD) in immediate postplacental insertion (IPPI). METHODS: 910 vaginal delivery women in Shanghai received a TCu 380A IUD inserted within 10 minutes after delivery of the placenta. Among them, 97.7% were primipara. The women were randomly divided into two groups: 470 cases in hand-insertion group and 440 in ring forceps-insertion group. Using life-tale method and X2 test, we compared the expulsion rates and other causes of removal after follow-up for 12 months in the two groups. RESULTS: The follow-up rate at 6 and 12 months were 95.16% and 92.64%, respectively. No uterine perforation and infection occurred in the 910 cases, and only one pregnancy in the hand-insertion group. Expulsions were the main reason for discontinuation. The 12-month gross cumulative expulsion rates were 15.86 and 15.88 per 100 women in the hand-insertion group and ring forceps-insertion group, respectively, and the removal rates due to bleeding and (or) pain were 2.11 and 1.57, respectively. No difference was statistically significant (P > 0.05). CONCLUSION: The two different insertion techniques do not significantly affect discontinuation rates in vaginal IPPI using the TCu 38)A IUD; the TCu 380A IUD appears to be suitable for postpartum insertion in Chinese women.

Adult↗
PubMed2005

Two independent retrotransposon insertions at the same site within the coding region of BTK.

Insertion of endogenous retrotransposon sequences accounts for approximately 0.2% of disease causing mutations. These insertions are mediated by the reverse transcriptase and endonuclease activity of long interspersed nucleotide (LINE-1) elements. The factors that control the target site selection in insertional mutagenesis are not well understood. In our analysis of 199 unrelated families with proven mutations in BTK, the gene responsible for X-linked agammaglobulinemia, we identified two families with retrotransposon insertions at exactly the same nucleotide within the coding region of BTK. Both insertions, an SVA element and an AluY sequence, occurred 12 bp before the end of exon 9. Both had the typical hallmarks of a retrotransposon insertion including target site duplication and a long poly A tail. The insertion site is flanked by AluSx sequences 1 kb upstream and 1 kb downstream and an unusual 60 bp sequence consisting of only As and Ts is located in intron 9, 60 bp downstream of the insertion. The occurrence of two retrotransposon sequences at exactly the same site suggests that this site is vulnerable to insertional mutagenesis. A better understanding of the factors that make this site vulnerable will shed light on the mechanisms of LINE-1 mediated insertional mutagenesis.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

Accommodation of insertion mutations on the surface and in the interior of staphylococcal nuclease.

Alignment of homologous amino acid sequences reveals that insertion mutations are fairly common in evolution. Hitherto, the structural consequences of insertion mutations on the surface and in the interior of proteins of known structures have received little attention. We report here the high-resolution X-ray crystal structures of 2 site-directed insertion mutants of staphylococcal nuclease. The structure of the first insertion mutant, in which 2 glycine residues were inserted on the protein surface in the amino-terminal beta-strand, has been solved to 1.70 A resolution and refined to a crystallographic R value of 0.182. The inserted residues are accommodated in a special 3-residue beta-bulge. A bridging water molecule in the newly created cavity satisfies the hydrogen bonding requirements of the beta-sheet by forming a bifurcated hydrogen bond to 1 beta-strand, and a single hydrogen bond to the other beta-strand. The second insertion mutant contains a single leucine residue inserted at the end of the third beta-strand. The structure was solved to 2.0 A resolution and refined to a final R value of 0.196. The insertion is accommodated in a register shift that changes the conformation of the flexible loop portion of the molecule, relaxing and widening the omega turn. This structural alteration results in changes in position and coordination of a bound calcium ion important for catalysis. These structures illustrate important differences in how amino acid insertions are accommodated: as localized bulges, and as extensive register shifts.

Amino Acid Sequence↗
PubMed2002

Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis.

A key component of a sound functional genomics infrastructure is the availability of a knockout mutant for every gene in the genome. A fruitful approach to systematically knockingout genes in the plant Arabidopsis thaliana has been the use of transferred-DNA (T-DNA) from Agrobacterium tumefaciens as an insertional mutagen. One of the assumptions underlying the use of T-DNA as a mutagen is that the insertion of these DNA elements into the Arabidopsis genome occurs at randomly selected locations. We have directly investigated the distribution of T-DNA insertions sites in populations of transformed Arabidopsis using two different approaches. To begin with, we utilized a polymerase chain reaction (PCR) procedure to systematically catalog the precise locations of all the T-DNA elements inserted within a 65 kb segment of chromosome IV. Of the 47 T-DNA insertions identified, 30% were found within the coding regions of genes. We also documented the insertion of T-DNA elements within the centromeric region of chromosome IV. In addition to these targeted T-DNA screens, we also mapped the genomic locations of 583 randomly chosen T-DNA elements by sequencing the genomic DNA flanking the insertion sites from individual T-DNA-transformed lines. 35% of these randomly chosen T-DNA insertions were located within the coding regions of genes. For comparison, coding sequences account for 44% of the Arabidopsis genome. Our results demonstrate that there is a small bias towards recovering T-DNA insertions within intergenic regions. However, this bias does not limit the utility of T-DNA as an effective insertional mutagen for use in reverse-genetic strategies.

Arabidopsis↗
PubMed1994

The effects of insertions on mammalian intrachromosomal recombination.

We examined the effects of insertion mutations on intrachromosomal recombination. A series of mouse L cell lines carrying mutant herpes simplex virus thymidine kinase (tk) heteroalleles was generated; these lines differed in the nature of their insertion mutations. In direct repeat lines with different large insertions in each gene, there was a 20-fold drop in gene conversion rate and only a five-fold drop in crossover rate relative to the analogous rates in lines with small insertions in each gene. Surprisingly, in direct repeat lines carrying the same large insertion in each gene, there was a larger drop in both types of recombination. When intrachromosomal recombination between inverted repeat tk genes with different large insertions was examined, we found that the rate of gene conversion dropped five-fold relative to small insertions, while the rate of crossing over was unaffected. The differential effects on conversion and crossing over imply that gene conversion is more sensitive to insertion mutation size. Finally, the fraction of gene conversions associated with a crossover increased from 2% for inverted repeats with small insertions to 18% for inverted repeats with large insertions. One interpretation of this finding is that during intrachromosomal recombination in mouse cells long conversion tracts are more often associated with crossing over.

Animals↗
PubMed2006

Sequential reorganization of beta-sheet topology by insertion of a single strand.

Insertions, duplications, and deletions of sequence segments are thought to be major evolutionary mechanisms that increase the structural and functional diversity of proteins. Alternative splicing, for example, is an intracellular editing mechanism that is thought to generate isoforms for 30%-50% of all human genes. Whereas the inserted sequences usually display only minor structural rearrangements at the insertion site, recent observations indicate that they may also cause more dramatic structural displacements of adjacent structures. In the present study we test how artificially inserted sequences change the structure of the beta-sheet region in T4 lysozyme. Copies of two different beta-strands were inserted into two different loops of the beta-sheet, and the structures were determined. Not surprisingly, one insert "loops out" at its insertion site and forms a new small beta-hairpin structure. Unexpectedly, however, the second insertion leads to displacement of adjacent strands and a sequential reorganization of the beta-sheet topology. Even though the insertions were performed at two different sites, looping out occurred at the C-terminal end of the same beta-strand. Reasons as to why a non-native sequence would be recruited to replace that which occurs in the native protein are discussed. Our results illustrate how sequence insertions can facilitate protein evolution through both local and nonlocal changes in structure.

Amino Acid Motifs↗
PubMed2004

A collection of 11 800 single-copy Ds transposon insertion lines in Arabidopsis.

More than 10 000 transposon-tagged lines were constructed by using the Activator (Ac)/Dissociation (Ds) system in order to collect insertional mutants as a useful resource for functional genomics of Arabidopsis. The flanking sequences of the Ds element in the 11 800 independent lines were determined by high-throughput analysis using a semi-automated method. The sequence data allowed us to map the unique insertion site on the Arabidopsis genome in each line. The Ds element of 7566 lines is inserted in or close to coding regions, potentially affecting the function of 5031 of 25 000 Arabidopsis genes. Half of the lines have Ds insertions on chromosome 1 (Chr. 1), in which donor lines have a donor site. In the other half, the Ds insertions are distributed throughout the other four chromosomes. The intrachromosomal distribution of Ds insertions varies with the donor lines. We found that there are hot spots for Ds transposition near the ends of every chromosome, and we found some statistical preference for Ds insertion targets at the nucleotide level. On the basis of systematic analysis of the Ds insertion sites in the 11 800 lines, we propose the use of Ds-tagged lines with a single insertion in annotated genes for systematic analysis of phenotypes (phenome analysis) in functional genomics. We have opened a searchable database of the insertion-site sequences and mutated genes (http://rarge.gsc.riken.go.jp/) and are depositing these lines in the RIKEN BioResource Center as available resources (http://www.brc.riken.go.jp/Eng/).

Arabidopsis↗
PubMed1995

Insert regions in domain X of the casein kinase II catalytic subunit.

Casein kinase II, cyclin-dependent kinases, and glycogen synthase kinase-3 are members of the protein kinase subfamily with a prominent insert in domain X of their catalytic subunit sequence. The function of the insert sequence in casein kinase II was investigated utilising synthetic peptides corresponding to the insert, cross-linking experiments, and the generation of casein kinase II insert region mutants. The mutation of basic residues (R276-->A, R278-->A, R281-->A, K277-->A) within the major insert sequence (PRFHDILQRHSRKRWERFVHSDNQHL, positions 265-290) did not affect alpha/beta subunit association, enzyme tetramerisation, thermal stability, and peptide (RRRDDDSDDD-NH2) phosphorylation. Similarly, replacement of residues 276-290 within the major insert with the corresponding residues from the cell-cycle kinase cyclin-dependent kinase 2 (CDK2) (FPKWKPGSLASHVKN) had no significant effect. The mutation of charged residues (H232-->A, H234-->A, D235-->A) within a nearby minor insert sequence (HGHDNY, positions 232-237), or replacement of residues 234-237 with the corresponding residues from CDK2 (DSEI) also did not affect alpha/beta subunit association and tetramerisation, but reduced enzyme thermal stability to more closely resemble the stability of the isolated alpha-subunit. In addition, mutations within the minor insert caused approximately a threefold increase in the apparent Km for peptide substrate. The results indicate that the major and minor inserts are not essential for alpha/beta subunit association, but the minor insert region influences substrate binding and thermal stability.

Amino Acid Sequence↗
PubMed1993

Insertion derivatives containing segments of up to 16 amino acids identify surface- and periplasm-exposed regions of the FhuA outer membrane receptor of Escherichia coli K-12.

The FhuA receptor in the outer membrane of Escherichia coli K-12 is involved in the uptake of ferrichrome, colicin M, and the antibiotic albomycin and in infection by phages T1, T5, and phi 80. Fragments of up to 16 amino acid residues were inserted into FhuA and used to determine FhuA active sites and FhuA topology in the outer membrane. For this purpose antibiotic resistance boxes flanked by symmetric polylinkers were inserted into fhuA and subsequently partially deleted. Additional in-frame insertions were generated by mutagenesis with transposon Tn1725. The 68 FhuA protein derivatives examined contained segments of 4, 8, 12, 16, and 22 additional amino acid residues at 34 different locations from residues 5 to 646 of the mature protein. Most of the FhuA derivatives were found in normal amounts in the outer membrane fraction. Half of these were fully active toward all ligands, demonstrating proper insertion into the outer membrane. Seven of the 12- and 16-amino-acid-insertion derivatives (at residues 378, 402, 405, 415, 417, 456, and 646) were active toward all of the ligands and could be cleaved by subtilisin in whole cells, suggesting a surface location of the extra loops at sites which did not affect FhuA function. Two mutants were sensitive to subtilisin (insertions at residues 511 and 321) but displayed a strongly reduced sensitivity to colicin M and to phages phi 80 and T1. Four of the insertion derivatives (at residues 162, 223, 369, and 531) were cleaved only in spheroplasts and probably form loops at the periplasmic side of the outer membrane. The number and size of the proteolytic fragments indicate cleavage at or close to the sites of insertion, which has been proved for five insertions by amino acid sequencing. Most mutants with functional defects were affected in their sensitivity to all ligands, yet frequently to different degrees. Some mutants showed a specifically altered sensitivity to a few ligands; for example, mutant 511-04 was partially resistant only to colicin M, mutant 241-04 was reduced in ferrichrome and albomycin uptake and showed a reduced colicin M sensitivity, and mutant 321-04 was fully resistant to phage T1 and partially resistant to phage phi 80. The altered residues define preferential binding sites for these ligands. Insertions of 4 to 16 residues at positions 69, 70, 402, 530, 564, and 572 resulted in strongly reduced amounts of FhuA in the outer membrane fraction, varying in function from fully active to inactive. These results provide the basis for a model of FhuA organization in the outer membrane.

Amino Acid Sequence↗
PubMed2001

Genomic stability of murine leukemia viruses containing insertions at the Env-3' untranslated region boundary.

Retroviruses containing inserts of exogenous sequences frequently eliminate the inserted sequences upon spread in susceptible cells. We have constructed replication-competent murine leukemia virus (MLV) vectors containing internal ribosome entry site (IRES)-transgene cassettes at the env-3' untranslated region boundary in order to examine the effects of insert sequence and size on the loss of inserts during viral replication. A virus containing an insertion of 1.6 kb replicated with greatly attenuated kinetics relative to wild-type virus and lost the inserted sequences in a single infection cycle. In contrast, MLVs containing inserts of 1.15 to 1.30 kb replicated with kinetics only slightly attenuated compared to wild-type MLV and exhibited much greater stability, maintaining their genomic integrity over multiple serial infection cycles. Eventually, multiple species of deletion mutants were detected simultaneously in later infection cycles; once detected, these variants rapidly dominated the population and thereafter appeared to be maintained at a relative equilibrium. Sequence analysis of these variants identified preferred sites of recombination in the parental viruses, including both short direct repeats and inverted repeats. One instance of insert deletion through recombination with an endogenous retrovirus was also observed. When specific sequences involved in these recombination events were eliminated, deletion variants still arose with the same kinetics upon virus passage and by apparently similar mechanisms, although at different locations in the vectors. Our results suggest that while lengthened, insert-containing genomes can be maintained over multiple replication cycles, preferential deletions resulting in loss of the inserted sequences confer a strong selective advantage.

3' Untranslated Regions↗