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PubMed1998

Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes.

A series of modular mini-transposon derivatives which permit the rapid cloning and mapping of the DNA flanking the minitransposon's site of insertion has been developed. The basic plasposon, named TnMod, consists of the Tn5 inverted repeats, a conditional origin of replication, rare restriction endonuclease multiple cloning sites, and exchangeable antibiotic resistance cassettes. The broad host range and low target DNA sequence specificity of the Tn5 transposase, in combination with the flexibility afforded by the modular arrangement of TnMod, result in a versatile tool for the mapping of insertional mutations and the rapid recovery of clones from gram-negative bacteria.

DNA Transposable Elements↗

PubMed1994

Cosmid cloning and restriction endonuclease mapping of the herpesvirus of turkeys (HVT) genome.

The genomic libraries of the herpesvirus of turkeys (HVT) presented to date do not include the entire genome. To construct a complete genomic library, HVT DNA was partially digested with Sau3A and inserted into the double cos site vector pcos2EMBL. The PstI maps derived from the partial Sau3A library demonstrate that the left region of the genome compared to the right area is overrepresented. Similar to the libraries of the HVT genome established earlier, a defined portion of the middle genomic region, however, is not contained in the partial Sau3A library. Cloning of HVT DNA in pcos2EMBL, employing BamHI for the partial digestion step, enabled to find the recombinant cosmid cBL267 which carries the BamHI-C and -D fragment as DNA insertion. As shown by Southern blot hybridization, the BamHI-C fragment ranges in a size to close the gap in the partial Sau3A library and thus guarantees the completeness of the genomic library of HVT which consists of seven overlapping cosmid clones (cBL1, cBL328, cBL11, cBL267, cBL27, cBL33, and cBL34).

Animals↗

PubMed1995

Insertion-site anatomy of the human menisci: gross, arthroscopic, and topographical anatomy as a basis for meniscal transplantation.

A cadaveric study was performed to determine the insertion-site anatomy of the human menisci, their topographical relationships to adjacent intra-articular structures, and which arthroscopic portal provides for optimal visualization of each insertion site. Fifteen fresh-frozen cadaver knees were studied (ages 48 to 63 years). Ten knees underwent arthroscopy using four standard arthroscopic portals. Visualization and placement of an arthroscopic guide over each meniscal horn insertion site was attempted through the four arthroscopic portals. Guide wires were drilled to mark horn insertions followed by a gross dissection to evaluate accuracy of the guide wire gross dissection to evaluate accuracy of the guide wire placement and to isolate meniscal horn insertion sites. Insertion sites were outlined and evaluated for size and topographical relationships to other intra-articular structures. Five additional knees were dissected free of all soft tissues except the tibial insertions of the meniscal roots and anterior cruciate ligament/posterior cruciate ligament. Each tibia was mounted in a jig and a digitizing system was used to record coordinates of points along the outline of each bony meniscal horn insertion site, the ACL tibial insertion, and the articular surface of each tibial plateau. The x, y, z coordinates for each point were calculated and loaded into a computer program allowing for surface area determination and computer-generated topographical maps to assess relative position of each specific insertion site. Placement of the arthroscope in the anterolateral portal allows optimal visualization and guide wire placement for both lateral meniscal horn insertion sites. Medial meniscal anterior and posterior horn insertion sites are best visualized with the arthroscope in the anteromedial and posteromedial portals respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthroscopy↗

PubMed2002

Monitoring arterial blood pressure: what you may not know.

Hemodynamic monitoring is a costly procedure, both materially and with regard to nursing time involved to ensure proper functioning of the monitoring system and correct interpretation of the data obtained. Dynamic response testing is the ideal method of confirming the ability of a monitoring system to accurately reproduce hemodynamic waveforms. MAP is a stable hemodynamic parameter, because it is least affected by monitoring method, catheter insertion site, the dynamic response characteristics of the catheter system, and wave reflection. MAP provides the best estimate of central aortic pressure and is the main hemodynamic parameter monitored by the neurohormonal system to control blood pressure. The superior informational value of MAP provides strong support for its preferred use in clinical practice, especially when use of vasoactive drugs is started or the dosages of these drugs are titrated. However, numerically satisfactory ABP or MAP values are not necessarily related to adequate peripheral tissue perfusion and organ system function. For optimal management of patients, data obtained from assessment tools such as hemodynamic monitoring devices must be integrated with information gained from clinical assessment of patients' status.

Blood Pressure↗

PubMed1994

Isolation of an allele of reeler by insertional mutagenesis.

Reeler (rl) is an autosomal recessive mutation that affects migration of postmitotic neurons in the mouse central nervous system. The reeler (rl/rl) mouse displays a disruption of laminar structures in both the cerebellum and the forebrain and it exhibits tremors, dystonia, and ataxia. The molecular basis of the reeler phenotype is unknown because the gene involved has not yet been identified. We report here the isolation and characterization of an allele of rl, reelertransgene (rltg). This allele was generated by the fortuitous insertion of a transgene, supfos (sf), into the mouse rl locus. Crosses between rl/+ and rltg/+ mice yielded offspring that exhibited the reeler phenotype, indicating that rl and rltg are allelic. We cloned the genomic sequences flanking the transgene insertion site from the rltg/rltg mouse genome. Chromosomal mapping studies revealed that the 5' flanking cellular sequence maps to a locus, D5Gmr1, that lies in a region of mouse chromosome 5 that also contains the rl locus. Southern blot analysis using a probe derived from the D5Gmr1 locus revealed no gross structural rearrangement in the rl locus. Thus, unlike the two rl alleles described previously, rltg provides a molecular probe that can now be used to identify and isolate the rl gene.

Alleles↗

PubMed1982

Physical and genetic localization of ilv regulatory sites in lambda ilv bacteriophages.

A set of nine lambda dilv phages were used to transduce bacterial recipients containing point mutations or deletions in the ilv genes located at 84 min on the Escherichia coli K-12 chromosome. This genetic analysis indicated that two phages carry the entire ilvGEDAC cluster; others carry the complete ilvC gene and, in addition, bacterial DNA that extends to a termination point between ilvA and ilvC, within ilvD, within ilvE, or within ilvG. DNA extracted from the lambda dilv phages was digested with EcoRI, HindIII, KpnI, PstI, SalI, and SmaI. The restriction maps revealed that these phages were generated after insertion at four distinct insertion sites downstream (clockwise) of ilvC. The physical relationships between the various phages were further examined by electron microscopic heteroduplex analysis. The physical maps of the phages thus generated were straightforward and in complete accord with the genetic data. No evidence for genetic rearrangements of ilv DNA in the phage was obtained, thus validating conclusions based on the use of these phages in previous and ongoing research projects. Bacterial cells with deletions of the ilv genes were made lysogenic with lambda dilv phage to examine the regulation of ilv genes present in the phage. The results confirm previous studies showing that one site for control by repression and derepression is upstream (counterclockwise) of ilvG. It was shown, in addition, that the activities of dihydroxy acid dehydrase and threonine deaminase were increased when the prototrophic lysogens were grown with 20 mM leucine. Since this increase was exhibited even when the ilvG-linked control region was not carried by the lambda dilv phage, additional control sites must be located within the ilvEDA region of the ilvGEDA transcription unit.

Amino Acids, Branched-Chain↗

PubMed1984

A cluster of five genes specifying the aerobactin iron uptake system of plasmid ColV-K30.

The genetic determinants for the aerobactin iron uptake system of plasmid ColV-K30, cloned as recombinant plasmid pABN1, were mapped by insertional inactivation using Tn1000 (gamma delta). Sites of insertion resulting in loss of aerobactin biosynthesis spanned ca. 5.5 kilobase pairs of cloned ColV-K30 DNA contiguous with a 2-kilobase-pair region in which transposon insertion resulted in loss of the outer membrane ferric-aerobactin receptor protein. Translation products of plasmid pABN1, and of subclones specifying siderophore biosynthesis alone or receptor activity alone, were analyzed by using the maxicell and minicell expression system. Four polypeptides (Mr = 62,000, 35,000, 45,000, and 50,000) are required for biosynthesis of aerobactin. A fifth product (Mr = 74,000) of plasmid pABN1 represents the outer membrane receptor protein. The linear order of genes for these polypeptides was determined by comparing translation products of a series of smaller derivative plasmids and of a number of mutant plasmids carrying Tn1000 at known locations.

Bacterial Outer Membrane Proteins↗

PubMed1995

Spatiotemporal relationships between a novel Drosophila stripe expressing gene and known segmentation genes by simultaneous visualization of transcript patterns.

We describe the cloning of the Drosophila transcription unit odd-striped (ost), which maps close to the P-element insertion site of the enhancer-trap line 05279. In order rapidly to gain information on its chromosomal localization, and to determine the spatial and temporal expression patterns of ost in whole-mount embryos, we established a double-label in situ hybridization protocol to localize two different DNA or RNA sequences simultaneously. The double-label in situ hybridization method involves digoxigenin- and biotin-labeled DNA probes that are processed to result in blue alkaline phosphatase and brown peroxidase reaction products, respectively. Using reference probes as internal standards, we show that the ost transcription unit is located within the cytogenetic band interval 89A1,2 on the right arm of the third chromosome, and that it exerts diagnostic segmentation gene expression patterns in the embryo. The ost transcripts are initially expressed in an anterior cap at the blastoderm stage, followed by a transient pair-rule gene expression pattern, which eventually changes into a segmental polarity gene pattern at gastrulation. Our results establish detailed spatial and temporal relationships between ost expression and the known patterns of the segmentation genes fushi tarazu and wingless, and demonstrate the potential of the double-label method with differently tagged DNA probes.

Animals↗

PubMed1995

Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR.

Thermal asymmetric interlaced (TAIL-) PCR is an efficient technique for amplifying insert ends from yeast artificial chromosome (YAC) and P1 clones. Highly specific amplification is achieved without resort to complex manipulations before or after PCR. The adaptation of this method for recovery and mapping of genomic sequences flanking T-DNA insertions in Arabidopsis thaliana is described. Insertion-specific products were amplified from 183 of 190 tested T-DNA insertion lines. Reconstruction experiments indicate that the technique can recover single-copy sequences from genomes as complex as common wheat (1.5 x 10(10) bp). RFLPs were screened using 122 unique flanking sequence probes, and the insertion sites of 26 T-DNA transgenic lines were determined on an RFLP map. These lines, whose mapped T-DNA insertions confer hygromycin resistance, can be used for fine-scale mapping of linked phenotypic loci.

Arabidopsis↗

PubMed1999

The fit-1 common integration locus in human and mouse is closely linked to MYB.

The fit-1 locus was originally identified as a common insertion site for feline leukemia virus (FeLV) in thymic lymphosarcomas induced by FeLV-myc recombinant viruses, suggesting that it harbors a gene that cooperates with Myc in T-cell leukemogenesis. We have previously mapped the fit-1 locus to feline Chromosome (Chr) B2. We have now identified conserved sequences that allow the mapping of the murine homolog using the European Interspecific Backcross (EUCIB). This shows that fit-1 is located on mouse Chr 10, 1cM proximal to Ahi-1, a murine retroviral integration locus that is closely linked to Myb. Moreover, the physical linkage to MYB is maintained in the human genome, as shown by cloning of the human homolog of fit-1 from a Chr 6 cosmid library and a series of overlapping PAC clones. Generation of a contig map around the human homolog of fit-1 reveals that it is approximately 100-kb upstream of MYB. In addition to fit-1 and Ahi-1, two other common insertion sites, Mis-2 and Mml-1, have also been mapped adjacent to Myb on mouse Chr 10. Previous analysis of tumors carrying insertions at fit-1, Mml-1, Mis-2 and Ahi-1 showed no obvious abnormalities in Myb expression. However, the cluster of viral insertion loci in this region suggests either the presence of a closely linked activation target or that subtle effects on Myb have been overlooked.

Animals↗

PubMed1986

Molecular cloning of a recA-like gene of Methylophilus methylotrophus and identification of its product.

A recombinant plasmid, pPF5, has been constructed which contains a 5.5-kb fragment of Methylophilus methylotrophus DNA inserted into pAT153, and which confers resistance to UV and mitomycin C (MC) upon Escherichia coli recA mutants. Hybridisation analysis indicates that sequence homology exists between the cloned DNA and a fragment of E. coli chromosomal DNA that contains the recA gene. Tn1000 insertions have been isolated within pPF5 that inactivate its ability to complement recA mutations, and the site of each insertion has been determined by restriction mapping. A 36-kDa protein is synthesised by pPF5 but not by any of the Tn1000 derivatives, indicating that this protein is the product of the gene responsible for the complementation. Comparison of the size of truncated polypeptides produced by selected pPF5::Tn1000 derivatives with the position of the insertion sites of the transposon, gave the direction of transcription of the M. methylotrophus recA+ gene. SOS proteins are induced in E. coli recA mutants harbouring pPF5 following MC treatment.

Cloning, Molecular↗

PubMed2002

Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient mice.

We have used large-scale insertional mutagenesis to identify functional landmarks relevant to cancer in the recently completed mouse genome sequence. We infected Cdkn2a(-/-) mice with Moloney murine leukemia virus (MoMuLV) to screen for loci that can participate in tumorigenesis in collaboration with loss of the Cdkn2a-encoded tumor suppressors p16INK4a and p19ARF. Insertional mutagenesis by the latent retrovirus was synergistic with loss of Cdkn2a expression, as indicated by a marked acceleration in the development of both myeloid and lymphoid tumors. We isolated 747 unique sequences flanking retroviral integration sites and mapped them against the mouse genome sequence databases from Celera and Ensembl. In addition to 17 insertions targeting gene loci known to be cancer-related, we identified a total of 37 new common insertion sites (CISs), of which 8 encode components of signaling pathways that are involved in cancer. The effectiveness of large-scale insertional mutagenesis in a sensitized genetic background is demonstrated by the preference for activation of MAP kinase signaling, collaborating with Cdkn2a loss in generating the lymphoid and myeloid tumors. Collectively, our results show that large-scale retroviral insertional mutagenesis in genetically predisposed mice is useful both as a system for identifying genes underlying cancer and as a genetic framework for the assignment of such genes to specific oncogenic pathways.

Animals↗

PubMed2001

Evolutionary dynamics of multiple group I introns in nuclear ribosomal RNA genes of endoparasitic fungi of the genus Cordyceps.

A large number of group I introns were discovered in coding regions of small and large subunits of nuclear ribosomal RNA genes (SSU rDNA and LSU rDNA) in ascomycetous fungi of the genus CORDYCEPS: From 28 representatives of the genus, we identified in total 69 group I introns which were inserted at any of four specific sites in SSU rDNA and four specific sites in LSU rDNA. These group I introns reached sizes of up to 510 bp, occurred in up to eight sites in the same organism, and belonged to either subgroup IB3 or subgroup IC1 based on their sequence and structure. Introns inserted at the same site were closely related to each other among Cordyceps fungi, whereas introns inserted at different sites were phylogenetically distinct even in the same species. Mapped on the host phylogeny, the group I introns were generally not restricted to a particular lineage, but, rather, widely and sporadically distributed among distinct lineages. When the phylogenetic relationships of introns inserted at the same site were compared with the phylogeny of their hosts, the topologies were generally significantly congruent to each other. From these results, the evolutionary dynamics of multiple group I introns in Cordyceps fungi was inferred as follows: (1) most of the group I introns were already present at the eight sites in SSU and LSU rDNAs of the ancestor of the genus Cordyceps; (2) the introns have principally been immobile and vertically transmitted throughout speciation and diversification of Cordyceps fungi, which resulted in the phylogenetic congruence between the introns at the same site and their hosts; (3) in the course of vertical transmission, the introns have repeatedly been lost in a number of lineages independently, which has led to the present sporadic phylogenetic distribution of the introns; and (4) a few acquisitions of new introns, presumably through horizontal transmission, were identified in the evolutionary history of the genus Cordyceps, while no transpositions were detected. Losses of group I introns in SSU rDNA have occurred at least 27 times in the evolutionary course of the 28 Cordyceps members.

Base Sequence↗

PubMed1990

An insertion of insect cell DNA in the 81-map-unit segment of Autographa californica nuclear polyhedrosis virus DNA.

In this report, a transposonlike insertion of Spodoptera frugiperda insect cell DNA was analyzed in single-plaque isolate E of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The 634-base-pair insertion is characterized by an 18-base-pair terminal inverted repeat and carries an EcoRI site. This additional EcoRI site in the 81-map-unit segment of the DNA of plaque isolate E of AcNPV explains the difference between the EcoRI restriction map of the DNA from this isolate and those of the virus stocks used in other laboratories. Except for this insertion, the nucleotide sequence at the site of insertion in the DNA of plaque isolate E is identical to that of AcNPV E2 (G. E. Smith and M. D. Summers, Virology 89:517-527, 1978). The cellular DNA insertion in the AcNPV genome is represented many times in the S. frugiperda cell genome but has no detectable homology with DNAs from species other than lepidopteran insects. In S. frugiperda cells, the transposonlike insertion sequences are transcribed into cytoplasmic RNA. The transcription of these sequences is initiated within the cellular insertion element. As reported previously (C. Oellig, B. Happ, T. Müller, and W. Doerfler, J. Virol. 61:3048-3057, 1987), in S. frugiperda cells infected with plaque isolate E of AcNPV, at least nine different size classes of AcNPV-specific RNAs are synthesized; in AcNPV E2-infected cells, similar size classes have been detected. The cellular insertion of plaque isolate E provides the initiation site for the synthesis of an additional RNA size class which is transcribed off viral DNA.

Animals↗

PubMed1992

Mutation and mapping of genes involved in production of the antibiotic TA in Myxococcus xanthus.

Transposition of TnV and Tn5lac into Myxococcus xanthus yielded 8,381 kanamycin-resistant mutants that were tested for antibiotic TA production. Twenty-four of the mutants were nonproducers of TA (less than 0.4 ng/ml), and 3 produced a higher level (2.5 micrograms/ml) than the parent strain (1.5 micrograms/ml). For most of the strains, there was 100% cotransduction between kanamycin resistance and the altered TA phenotype. Southern blot analysis of restriction digests of the mutant DNA indicated that the transposons were inserted at different sites on the M. xanthus chromosome. The TA genes were mapped by cotransduction between pairs of mutants following replacement of the initial insert of one of the pair with the tetracycline resistance transposon Tn5-132. Nine of the 13 nonproducers tested were linked over a 36-kb stretch of the chromosome. There was no linkage between one of the overproducers and any of the nonproducers tested.

Anti-Bacterial Agents↗

PubMed1989

Use of yeast artificial chromosome clones for mapping and walking within human chromosome segment 18q21.3.

Well-characterized large genomic clones obtained from yeast artificial chromosome (YAC) libraries provide the framework to localize genes and approach genetic disease. We developed universally applicable approaches to establish authenticity, localize and orient internal genes, map restriction sites, and rescue the distal ends of large human genomic DNA inserts. We selected human chromosome segment 18q21.3 as a model system. Molecular cloning of this segment was initiated by characterizing three plasminogen activator inhibitor type 2 (PAI-2) clones [290, 180, and 60 kilobases (kb)] isolated from a YAC library. Comparison of YAC and bacteriophage lambda genomic DNA clones confirmed the fidelity of the PAI-2 locus. Detailed rare cutting restriction maps were generated by ramped contour-clamped homogeneous electric field electrophoresis. The PAI-2 locus was located and oriented within the YACs, which span a distance 70 kb 5' to 220 kb 3' of PAI-2. Moreover, both left and right ends of the YAC genomic DNA inserts were rescued by amplifying circularized cloning sites with an inverted form of the polymerase chain reaction. These unique terminal genomic DNA fragments were used to rescreen the YAC library and isolate overlapping clones that extend the map. These approaches will enable neighboring loci to be definitively linked and establish the feasibility of using YAC technology to clone and map chromosomal segments.

Base Sequence↗

PubMed1991

Mapping catalytically important regions of an enzyme using two-codon insertion mutagenesis: a case study correlating beta-lactamase mutants with the three-dimensional structure.

Two-codon insertion mutants throughout the beta-lactamase (Bla)-encoding gene were characterized. Second site revertants of various mutants were isolated, mapped and sequenced. The activity of the mutants and the ability to get revertants showed a positive correlation with increasing distance from the active site, based on the three-dimensional structure of Bla. This observation is discussed as it may pertain to the generalized use of two-codon insertion mutagenesis in mapping important catalytic regions in enzymes.

Amino Acid Sequence↗

PubMed1983

Amelioration of the ultraviolet sensitivity of an Escherichia coli recA mutant in the dark by photoreactivating enzyme.

When a recA strain of Escherichia coli is transformed with a multicopy plasmid, pKY1, carrying the phr gene of E. coli, its extreme ultraviolet sensitivity is decreased. Derivatives of pKY1 were prepared in which the phr gene was inactivated by inserting transposon Tn1000. None of the 20 phr- derivatives decreased the UV sensitivity of the recA strain. In an analogous experiment, we obtained 11 derivatives which failed to decrease the UV sensitivity of the recA strain. None of them complemented phr strain. Furthermore, TN1000 insertion sites in both types of derivatives were mapped in the same region of the plasmid. From these observations, we propose that the E. coli phr gene product has repair activity in the dark.

Bacterial Proteins↗