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At least 19 recordsLinked to original sources
PubMed1990

Insertional mutagenesis of Listeria monocytogenes with a novel Tn917 derivative that allows direct cloning of DNA flanking transposon insertions.

To carry out efficient insertional mutagenesis in Listeria monocytogenes and to facilitate the characterization of disrupted genes, two novel derivatives of Tn917 were constructed, Tn917-LTV1 and Tn917-LTV3. The derivatives (i) transpose at a significantly elevated frequency, (ii) generate transcriptional lacZ fusions when inserted into a chromosomal gene in the appropriate orientation, and (iii) allow the rapid cloning in Escherichia coli of chromosomal DNA flanking transposon insertions. The rapid cloning of DNA flanking insertions is possible because the transposon derivatives carry ColE1 replication functions, a cluster of polylinker cloning sites, and antibiotic resistance genes selectable in E. coli (bla in the case of Tn917-LTV1; neo and ble in the case of Tn917-LTV3). The enhanced transposition frequency of Tn917-LTV1 and Tn917-LTV3 (about 100-fold in Bacillus subtilis) is believed to be due to the fortuitous placement of vector-derived promoters upstream from the Tn917 transposase gene. In L. monocytogenes, Tn917-LTV3 transposed at a frequency of 8 x 10(-4) when introduced on a pE194Ts-derived vector and generated at least eight different auxotrophic mutations. Two nonhemolytic insertion mutants of L. monocytogenes were isolated, and DNA flanking the transposon insertions was cloned directly into E. coli, making use of the ColE1 rep functions and neo gene carried by Tn917-LTV3. Both insertions were shown to be within hlyA, the L. monocytogenes hemolysin structural gene. Although Tn917-LTV1 and Tn917-LTV3 were constructed specifically for genetic analysis of L. monocytogenes, their enhanced transposition frequency and convenience for cloning of DNA adjacent to sites of insertions make them the transposon derivatives of choice for insertional mutagenesis in any gram-positive bacteria that support replication of pE194Ts.

Bacillus subtilis↗
PubMed1991

A new type of insertion mutation in monkey cells: insertion accompanied by long target site duplication.

We have developed a system for the detection of a new type of insertion mutation in mammalian cells. We have used a shuttle vector, plasmid pNK1, which contains the SV40 and pBR322 replication origins, and ApR, galK, and neoR genes. This plasmid was introduced into monkey COS1 cells, allowed to replicate, and then recovered plasmids were reintroduced into Escherichia coli HB101 to detect insertion mutations in the galK gene. We selected galK- KmR ApR mutants in order to eliminate galK- KmS deletion mutants. Insertion mutations in the plasmids recovered were then screened by agarose gel electrophoresis. Finally, insertion mutants that had the following characteristics were selected. First, they had the ability to produce gal+ revertants caused by the precise excision of inserted DNA in E. coli, implying that they had a target site duplication on both sides of the insertion. Second, they contained some repetitive sequence(s) as judged by hybridization with a bulk monkey DNA probe. Nucleotide sequence analysis of one of the mutants, 15K-1, showed that it contained alpha-satellite sequences within the coding region of the galK gene. It contained 13 1/2 tandem repeat units of alpha-satellite sequence and was flanked by a 64 bp target site duplication, indicating that the alpha-satellite sequence had been translocated from the monkey genome into the plasmid by illegitimate recombination. Another insertion mutant, N11-1, contained an 11 kb insert which included an unknown repetitive sequence that was also flanked by a target site duplication of 353 bp.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Mapping of insertion elements IS1, IS2 and IS3 on the Escherichia coli K-12 chromosome. Role of the insertion elements in formation of Hfrs and F' factors and in rearrangement of bacterial chromosomes.

The chromosome of an Escherichia coli K-12 strain W3110 contains seven copies of insertion element IS1, 12 copies of IS2 and six copies of IS3. We determined the approximate locations of six copies of IS1 (named is1A to is1F), ten copies of IS2 (named is2A to is2J), and five copies of IS3 (named is3A to is3E) on the W3110 chromosome by plaque hybridization using the "mini-set" of the lambda phage library that includes 476 clones carrying chromosomal segments that cover the W3110 chromosome almost entirely. Cleavage maps of the W3110 chromosome and cleavage analysis of phage DNAs carrying insertion elements allowed us to assign more precise locations to most of the insertion elements and to determine their orientations. Insertion elements were distributed randomly along the W3110 chromosome in one or other orientation. Several of these were located at the same positions on the chromosome of another E. coli K-12 strain, JE5519, and they were assumed to be the original complement of insertion elements in E. coli K-12 wild-type. Locations and orientations of such insertion elements were correlated well with Hfr points of origin and with crossover points for excision of some F' factors derived from several Hfrs. Insertion elements may be involved also in rearrangement of bacterial chromosomes.

Chromosomes, Bacterial↗
PubMed1991

Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains.

Most strains of the Mycobacterium tuberculosis complex carry multiple copies of an IS3-like element, and these strains are highly polymorphic with regard to the site of integration in the chromosome. In contrast, Mycobacterium bovis BCG contains a single copy of the insertion element, and in all strains this copy is integrated at the same site in the chromosome. In this study, we determined the sequence of the single-copy insertion element from M. bovis BCG, IS987, and its flanking regions. The analysis of IS987 revealed that this element was virtually identical to the sequence of IS986 from M. tuberculosis. IS987 is located in a region containing direct repeats (DRs). The cloned flanking regions contained 20 virtually identical DRs of 36 bp, each separated by 35 to 41 bp of spacer DNA. Analysis of chromosomal DNA by the polymerase chain reaction revealed the presence of a cluster of 49 DRs, and IS987 is inserted in the 30th DR. Furthermore, the DR sequences were found to occur only in species of the M. tuberculosis complex and not in nine other mycobacterial species tested. Analysis of 14 M. tuberculosis strains revealed the presence of one insertion sequence element in the DR-containing region of eight strains, two insertion sequence elements were located in the DR region of five strains, and one strain did not contain an insertion sequence element in this region. Additionally, the DR-containing regions of these 14 M. tuberculosis strains were polymorphic in length and composition. We conclude that the DR cluster is a specific, hot-spot region for integration of insertion elements in the chromosome of M. tuberculosis complex strains.

Amino Acid Sequence↗
PubMed1990

The developmental morphology of a "periosteal" ligament insertion: growth and maturation of the tibial insertion of the rabbit medial collateral ligament.

The structural properties of ligament insertions change dramatically during growth and maturation, but little is known about their developmental anatomy. This study describes and quantifies changes in the gross and microscopic anatomy of the tibial insertion of the rabbit medial collateral ligament (MCL) during development and at skeletal maturity. Eighty animals were used for growth and descriptive studies. From this group, 27 animals, ranging in age from 1 to 24 months, were injected with fluorescent bone markers and their tibial insertions were processed undecalcified for histology. Sections were examined by polarized light and fluorescence microscopy to identify matrix and cells and to quantify mineral formation. Results showed that animals achieved histological skeletal maturity between 9 and 12 months of age. Body weights were a poor index of skeletal maturity. The tibial insertion was composed of five tissue layers, which changed proportions during growth and maturation. In immature animals, MCL fibers entered the periosteum; in older animals, MCL fibers were cemented to the tibia by advancing mineral. The tibial attachment of the MCL was thus transferred from the periosteum to the cortex during growth, suggesting that the term "periosteal insertion" is imprecise in adults. The hypothesis is put forward that these structural changes account for the reported increase in tensile failure of this insertion near skeletal maturity.

Aging↗
PubMed1988

Comparisons of host cell DNA insertions and altered transcription at the site of insertions in few polyhedra bacilovirus mutants.

Few polyhedgra (FP) mutants of Autographa californica nuclear polyhedrosis virus (AcNPV) and the closely related strain Galleria mellonella (Gm)NPV have been reported which contain Trichoplusia ni host cell DNA sequences inserted into the viral genome between map units 35.0 and 37.7. New FP mutants are described with alterations of the HindIII-I restriction enzyme fragment (33.8 to 37.7 map units) of AcNPV, either deletions of viral DNA sequences or insertions of spodoptera frugiperda host cell DNA sequences. S. frugiperda DNA insertions from FP mutants were compared to T. ni host DNA insertions from FP mutants previously isolated (M.J. Fraser, G.E. Smith, and M.D. Summers (1983) J. Virol. 47, 287-300). Two host cell DNA sequences isolated from FP mutants, one of T. ni origin and one from S. frugiperda DNA, were transcribed in infected cells. Deletions of viral DNA sequences and insertions of host DNA sequences produce altered transcripts at the site of mutation as determined by both Northern and S1 nuclease analysis. Cell-free translation of cRNAs transcribed from wild-type viral DNA revealed an open reading frame coding for a 25-kDa protein at the site where host cell DNA insertions have been mapped. This was the same size as an infected-cell protein missing from most FP mutants examined.

DNA Transposable Elements↗
PubMed1985

Comparison of intramenstrual IUD insertion with insertion following menstrual regulation.

To evaluate the use-effectiveness and safety of IUD insertion immediately after menstrual regulation (MR) for delayed menses, a Lippes Loop D (LLD) intrauterine device was inserted in each of 100 consecutive clients at the University College Hospital, Ibadan, immediately after menstrual regulation. Pertinent event rates after 12 months of use were compared with those of 100 consecutive women who had the LLD inserted during menstruation. The cumulative net expulsion rate after 12 months of use was 8% for the study group and 4% for the controls. The overall rate of removals was 15% for the study group and 16% for the control group. None of the observed differences was statistically significant. The continuation rates at 12 months were comparable for both groups (78% and 80%, respectively). There were no accidental pregnancies during the study period. The authors suggest that IUD insertion immediately after menstrual regulation is as effective and safe as intramenstrual insertion, provided prophylactic antibiotics are given.

Adult↗
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↗
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↗
PubMed1979

Mu insertion duplicates a 5 base pair sequence at the host inserted site.

Nucleotide sequences were analyzed across the two ends of lysogenic Mu DNA. These ends were cloned separately in lambdapMu hybrid particles that derived from a single Mu lysogen in the lac Z part of lambdaplac5. The obtained data imply that Mu lysogenization was associated with the duplication of 5 base pairs present in lac DNA at the Mu insertion site. As a result of this duplication, Mu DNA is flanked by two copies of five identical base pairs oriented as direct repeats. A similar conclusion has been obtained independently by other investigators with the use of a different Mu lysogen (D. Kamp and R. Kahmann, personal communication). Thus Mu insertion seems to have a striking similarity to typical IS-mediated insertions that were found to be associated with a short DNA duplication at the target site.

Base Sequence↗
PubMed1989

Somatic reversion of P transposable element insertion mutations in the singed locus of Drosophila melanogaster requiring specific P insertions and a trans- acting factor.

Destabilization in somatic cells of P-element insertions in the X-linked singed gene of Drosophila melanogaster has been studied. We have shown that some but not all unstable P-element insertions in singed can form mosaics. The cause of this variation is not clear from studies of the restriction maps of the mutations tested. The transposable element movements occur early in development and require, in addition to an appropriate P-element insertion in singed, a trans-acting maternal effect component. Movements appear to occur preferentially in attached-X stocks. However, the maternal effect component maps to the central region of chromosome 2.

Animals↗
PubMed1987

The release of insoluble antibiotics from collagen ocular inserts in vitro and their insertion into the conjunctival sac of cattle.

The release rate of procaine penicillin, erythromycin and erythromycin estolate from soluble and insoluble collagen films was investigated in vitro to develop an ocular insert for the treatment of infectious bovine keratoconjunctivitis. The release rate and duration of release varied according to the selection of antibiotic and vehicle. The combination of erythromycin estolate and soluble collagen produced the most sustained drug-delivery system. However, due to the inappropriate physical properties of collagen and poor retention of ocular inserts, it was considered that the development of an antibiotic-impregnated collagen ocular insert requires further investigation.

Animals↗
PubMed1989

Specificity of insertion of IS91, an insertion sequence present in alpha-haemolysin plasmids of Escherichia coli.

We have determined the DNA sequences of eight different insertions of IS91 in a specifically engineered recipient plasmid of known DNA sequence (pSU300). The sequences at the termini of IS91 are 5'-CGAGTAGG...CCTATCGAT. IS91 inserts specifically 5' to either one of the tetranucleotides 5'-GAAC or 5'-CAAG, and always in the same relative orientation with respect to the sequence of the target. Except in one special case, no duplications of the recipient DNA were produced at the site of insertion.

Bacterial Proteins↗
PubMed1991

[Function of the InsA gene in the IS1 element of the Tn9' transposon: influence of oligonucleotide inserts in the InsA gene on formation of simple insertions and plasmid cointegrates].

To elucidate the role of the insA reading frame in transposition of the IS1 element of the Tn9' transposon, the derivatives of plasmids pUC19::Tn9' and pUC19::IS1 have been obtained using oligonucleotide inserts of the length equal or exceeding 9 bp and equal to 10 bp. The ability of mutant variants of the Tn9' transposon and the IS1 element to form simple insertions and plasmid cointegrates was studied. To this end, experiments were performed on mobilization of the derivatives of pUC19 containing mutant variants of the IS1 element and Tn9' as well as of the plasmids pUC19::Tn9' by the conjugative plasmid pRP3.1. According to the data obtained, mutations (inserts) in the insA gene have no influence on the frequency of transposition of the IS1 element and Tn9' from the plasmid pUC19 to pRP3.1. At the same time, the frequency of transposition events of mutant variants of Tn9' from the plasmid pRP3.1 to pBR322 is more than 10 times lower in comparison with the wild type transposon. The data obtained are in accordance with the assumption that the insA gene is not essential for transposition. A hypothesis is put forward explaining the role of the insA gene product in the process of bringing together short inverted repeats of the IS1, which are the sites for the transposase to be recognized at first stages of transposition.

DNA Transposable Elements↗
PubMed1991

Insertion of diphtheria toxin B-fragment into the plasma membrane at low pH. Characterization and topology of inserted regions.

When the enzymatically active A-fragment of diphtheria toxin is translocated to the cytosol, the B-fragment inserts into the membrane in such a way that a 25-kDa polypeptide becomes shielded from proteases added to the external medium. We have attempted to determine the boundaries of this polypeptide within the toxin B-fragment as well as the topology of the B-fragment in the membrane. Chemical cleavage of the 25-kDa polypeptide with hydroxylamine and o-iodosobenzoic acid yielded fragments of sizes indicating that the 25-kDa polypeptide starts at residue approximately 300 and extends to the COOH-terminal end. Experiments where the toxin was labeled with [35S]cysteine at distinct positions of the B-fragment supported this conclusion. Treatment of cells with inserted B-fragment with L-1-tosyl-amido-2-phenylethyl chloromethyl ketone-treated trypsin and with V8 protease from Staphylococcus aureus yielded protected 27- and 30-kDa fragments in addition to 25 kDa, indicating that the region 240-264 is also at the outside. The topology of the inserted B-fragment is discussed.

Amino Acid Sequence↗
PubMed1987

'Bowing' forces with IUD inserters in vitro: relevance to difficult IUD insertions.

The inserter tubes of the more modern copper bearing IUDs are flexible enough to give ('bow') when there is obstruction to the device. The MLCu 250, Copper 7 and Nova T inserters will bow considerably when forces of 1-3 N are exerted and the device obstructed up to 0.75 cm from its proximal end. This suggests that these devices are unlikely to cause cervical damage where their passage into the uterine cavity is impeded by cervical factors, provided the insertion attempt is discontinued when bowing of more than 2 cm off-centre is reached. However this does not apply to obstructions past the internal cervical os. In research circumstances great care should be used as it rapidly becomes possible to exert enough force to produce damage to the uterine muscle.

Equipment Failure↗
PubMed1987

Escherichia coli integration host factor binds specifically to the ends of the insertion sequence IS1 and to its major insertion hot-spot in pBR322.

We report here that the ends of IS1 are bound and protected in vitro by the heterodimeric protein integration host factor (IHF). Under identical conditions, RNA polymerase binds to one of these ends (IRL) and protects a region that includes the sequences protected by IHF. Other potential sites within IS1, identified by their homology to the apparent consensus sequence, are not protected. Footprinting analysis of deletion derivatives of the ends demonstrates a correspondence between the ability of the end sequence to bind IHF and its ability to function as an end in transposition. Nonetheless, some transposition occurs in IHF- cells, indicating that IHF is not an essential component of the transposition apparatus. IHF also binds and protects four closely spaced regions within the major hot-spot for insertion of IS1 in the plasmid pBR322. This striking correlation of hot-spot and IHF-binding sites suggests a possible role for IHF in IS1 insertion specificity.

Bacterial Proteins↗