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PubMed1989

Site-specific integration in Streptomyces ambofaciens: localization of integration functions in S. ambofaciens plasmid pSAM2.

In Streptomyces ambofaciens ATCC 15154, an 11.1-kilobase element, pSAM2, exists as a single integrated copy in the chromosome. In S. ambofaciens 3212 (a derivative of ATCC 15154), pSAM2 exists as a free, circular plasmid as well as an integrated element. BclI fragments from the free form of pSAM2 were cloned into an Escherichia coli plasmid vector. By using gene transplacement methods, the chromosomally integrated form of pSAM2 was marked with a gene coding for apramycin resistance. This enabled us to isolate both a segregant that had lost the integrated pSAM2 element and a cosmid clone containing integrated pSAM2 along with the flanking chromosomal sequences. One of the BclI fragments derived from free pSAM2 was shown to contain all the plasmid-specified information required to direct site-specific recombination in a derivative of S. ambofaciens lacking the resident pSAM2 element as well as in a number of other Streptomyces strains. The attachment sites used by the plasmid and the chromosome in site-specific recombination and the junctions created after integration were cloned and sequenced. Certain structural features in common with other integrating elements in actinomycetes were noted.

Base Sequence↗
PubMed1993

Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus.

Several pGEM5- and pUC19-derived plasmids containing a selectable erythromycin resistance marker were integrated into the chromosome of Streptococcus thermophilus at the loci of the lactose-metabolizing genes. Integration occurred via homologous recombination and resulted in cointegrates between plasmid and genome, flanked by the homologous DNA used for integration. Selective pressure on the plasmid-located erythromycin resistance gene resulted in multiple amplifications of the integrated plasmid. Release of this selective pressure, however, gave way to homologous resolution of the cointegrate structures. By integration and subsequent resolution, we were able to replace the chromosomal lacZ gene with a modified copy carrying an in vitro-generated deletion. In the same way, we integrated a promoterless chloramphenicol acetyltransferase (cat) gene between the chromosomal lacS and lacZ genes of the lactose operon. The inserted cat gene became a functional part of the operon and was expressed and regulated accordingly. Selective pressure on the essential lacS and lacZ genes under normal growth conditions in milk ensures the maintenance and expression of the integrated gene. As there are only minimal repeated DNA sequences (an NdeI site) flanking the inserted cat gene, it was stably maintained even in the absence of lactose, i.e., when grown on sucrose or glucose. The methodology represents a stable system in which to express and regulate foreign genes in S. thermophilus, which could qualify in the future for an application with food.

Base Sequence↗
PubMed1980

Patterns of viral DNA integration in cells transformed by wild type or DNA-binding protein mutants of adenovirus type 5 and effect of chemical carcinogens on integration.

The integration pattern of viral DNA was studied in a number of cell lines transformed by wild-type adenovirus type 5 (Ad5 WT) and two mutants of the DNA-binding protein gene, H5ts125 and H5ts107. The effect of chemical carcinogens on the integration of viral DNA was also investigated. Liquid hybridization (C(0)t) analyses showed that rat embryo cells transformed by Ad5 WT usually contained only the left-hand end of the viral genome, whereas cell lines transformed by H5ts125 or H5ts107 at either the semipermissive (36 degrees C) or nonpermissive (39.5 degrees C) temperature often contained one to five copies of all or most of the entire adenovirus genome. The arrangement of the integrated adenovirus DNA sequences was determined by cleavage of transformed cell DNA with restriction endonucleases XbaI, EcoRI, or HindIII followed by transfer of separated fragments to nitrocellulose paper and hybridization according to the technique of E. M. Southern (J. Mol. Biol. 98: 503-517, 1975). It was found that the adenovirus genome is integrated as a linear sequence covalently linked to host cell DNA; that the viral DNA is integrated into different host DNA sequences in each cell line studied; that in cell lines that contain multiple copies of the Ad5 genome the viral DNA sequences can be integrated in a single set of host cell DNA sequences and not as concatemers; and that chemical carcinogens do not alter the extent or pattern of viral DNA integration.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1993

Conceptualizing and measuring integration: findings from the health systems integration study.

Given the pressures for health care reform, interest in the concept of integrated or organized delivery systems as a means to offer more coordinated cost-effective care is growing. This article has two primary objectives: (1) to clarify the different types of integration associated with the notion of an organized delivery system, and (2) to share the results from an ongoing study of 12 organized delivery systems. The findings indicate a moderate level of integration overall, particularly in the areas of culture, financial planning, and strategic planning. The study found that corporate staff respondents perceive their systems to be more integrated and effective than do operating unit managers, and that some functional integration areas are positively associated with both physician-system and clinical integration that, in turn, are positively related to each other. Overall, perceived integration was found to be positively associated with perceived effectiveness.

Administrative Personnel↗
PubMed2001

HIV DNA integration during cell-to-cell transmission of infection: evidence for partially integrated DNA structures in acutely infected cells.

A one-step cell-to-cell transmission model of human immunodeficiency virus (HIV) infection was used to study viral DNA integration in the early phase of viral replication. Co-culturing H3B cells as virus donors with CD4+ Hut78 recipient cells in a ratio of 1:4 produced a synchronous, one-step viral infection with de novo synthesis of unintegrated HIV DNA within 4 h p.i., which subsequently integrates in the host genomic DNA to form provirus. To study the kinetics of viral DNA integration, cellular chromosomal DNA was isolated at different times after co-culturing and extensive electrophoresis was used to remove residual unintegrated viral DNA. Removal of contaminating, unintegrated viral DNA in the purified chromosomal DNA fraction was confirmed by various experiments. When purified chromosomal DNA (free of contaminating unintegrated viral DNA)--from the mix of acutely infected cells--was digested with restriction enzymes KpnI, BamHI or PstI and analysed by Southern blot hybridization, integration of viral DNA into chromosomal DNA was first observed at 8 h p.i. and was essentially complete by 72 h p.i. In addition, evidence was found for a relatively stable, partially integrated HIV DNA structure within the chromosomal DNA, that was first detectable at 8 h p.i. and did not become fully integrated until 72 hours post infection.

Blotting, Southern↗
PubMed2005

The SET domain protein Metnase mediates foreign DNA integration and links integration to nonhomologous end-joining repair.

The molecular mechanism by which foreign DNA integrates into the human genome is poorly understood yet critical to many disease processes, including retroviral infection and carcinogenesis, and to gene therapy. We hypothesized that the mechanism of genomic integration may be similar to transposition in lower organisms. We identified a protein, termed Metnase, that has a SET domain and a transposase/nuclease domain. Metnase methylates histone H3 lysines 4 and 36, which are associated with open chromatin. Metnase increases resistance to ionizing radiation and increases nonhomologous end-joining repair of DNA doublestrand breaks. Most significantly, Metnase promotes integration of exogenous DNA into the genomes of host cells. Therefore, Metnase is a nonhomologous end-joining repair protein that regulates genomic integration of exogenous DNA and establishes a relationship among histone modification, DNA repair, and integration. The data suggest a model wherein Metnase promotes integration of exogenous DNA by opening chromatin and facilitating joining of DNA ends. This study demonstrates that eukaryotic transposase domains can have important cell functions beyond transposition of genetic elements.

Amino Acid Sequence↗
PubMed1998

Site-specific integration of bacteriophage VWB genome into Streptomyces venezuelae and construction of a VWB-based integrative vector.

The temperate bacteriophage VWB integrates into the chromosome of Streptomyces venezuelae ETH14630 via site-specific integration. Following recombination of the VWB attP region with the chromosomal attB sequence, the host-phage junctions attL and attR are formed. Nucleotide sequence analysis of attP, attB, attL and attR revealed a 45 bp common core sequence. In attB this 45 bp sequence consists of the 3' end of a putative tRNA Arg(AGG) gene with a 3'-terminal CCA sequence which is typical for prokaryotic tRNAs. Phage DNA integration restores the putative tRNA Arg(AGG) gene in attL. However, following recombination the CCA sequence is missing as is the case for most Streptomyces tRNA genes described so far. Adjacent to VWB attP, an ORF encoding a 427 aa protein was detected. The C-terminal region of this protein shows high similarity to the conserved C-terminal domain of site-specific recombinases belonging to the integrase family. To prove the functionality of this putative integrase gene (int), an integrative vector pKT02 was constructed. This vector consists of a 2.3 kb HindIII-SphI restriction fragment of VWB DNA containing attP and int cloned in a non-replicative Escherichia coli vector carrying a thiostrepton-resistance (tsr) gene. Integration of pKT02 was obtained after transformation of Streptomyces venezuelae ETH14630 and Streptomyces lividans TK24 protoplasts. This vector will thus be useful for a number of additional Streptomyces species in which a suitable tRNA gene can be functional as integration site.

Amino Acid Sequence↗
PubMed1995

Network integration: the impact of integrated delivery systems. Roundtable discussion.

One of the hottest, most competitive segments of the healthcare IS industry encompasses integration tools and systems for linking divergent, best-of-breed systems. As firms such as Healthcare Communications, HUBLink, Oacis, Software Technologies Corp., Century Analysis and Eagle Innovations vie for new contracts, CIOs and IS directors wonder about the strategic advantages of single-vendor solutions vs. the best-of-breed approach. This dynamic has driven system acquisitions for several years. More recently, 33 percent of respondents to the 1995 HIMSS/HP Survey cited integration across separate facilities as their No. 1 priority for the next two years. Clearly the emergence of integrated delivery systems and providers of continuums of care--where the cost to replace the information technologies of acquired or partnered entities remains prohibitive--will cause the market for interface engines and other integration tools to continue expanding. Following are the perceptions of a few experts on the topic. They were asked to answer two questions: 1) What are the toughest challenges faced when integrating an integrated delivery system? and 2) What are the current best practices to solving this challenge?

Comprehensive Health Care↗
PubMedDate not supplied

Are provider integrated delivery systems the final step to integration?

Provider integrated delivery systems are not the final step to full-fledged integration, states author R. Daniel King, president of Medi-Call of St. Louis Inc. While a PIDS is on the way, true interdependent integration takes place when the community joins with the integrated system to represent all phases of the health care continuum and assumes some of the responsibility for planning. These community integrated delivery systems (CIDS) should be the aim of integration.

Community Health Planning↗
PubMed2000

Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains.

An improved method for integration of exogenous DNA fragments at a defined site within the genome of Pseudomonas aeruginosa was developed. The method relies on two integration-proficient vectors, mini-CTX1 and mini-CTX2. These two vectors contain (1) a tetracycline (tet) selectable marker, (2) an oriT for conjugation-mediated plasmid transfer, (3) the pMB1-derived origin of replication, (4) a modified φCTX integrase (int) gene, (5) a versatile multiple cloning site (MCS) flanked by T4 transcriptional termination sequences (Omega elements), and (6) the φCTX attachment site. The MCS and Omega elements are flanked by yeast Flp recombinase target sites that allow in vivo excision of unwanted plasmid backbone sequences, including tet and int, from the genome of integrants by Flp recombinase. In the mini-CTX2 vector int transcription is driven from the strong trc promoter, which is regulated by the Lac repressor that is encoded by lacI(q) also contained on the plasmid. Upon conjugal transfer, mini-CTX1 and mini-CTX2 integrated at frequencies of 10(-8) and 10(-7), respectively. The usefulness of the integration vectors for gene fusion analyses was demonstrated by chromosomal insertion of autoinducer (AI)-regulated lasB-lacZ and rhlA-lacZ fusions into wild-type and AI synthase mutants. In wild-type, the fusions responded in a cell density-dependent manner and expression of both fusions was either greatly reduced or abolished in AI synthase mutants. Finally, an expression cassette containing the T7 polymerase gene under Lac repressor control was constructed, integrated into the P. aeruginosa chromosome, and used to express the hexahistidine-tagged P. aeruginosa AI synthase RhlI.

Artificial Gene Fusion↗
PubMed2005

Osmotic tolerance of mouse spermatozoa from various genetic backgrounds: acrosome integrity, membrane integrity, and maintenance of motility.

All cells have an intrinsic biophysical property related to their ability to undergo osmotically driven volume changes. This project is of fundamental importance to our understanding of the basic cryobiology of mouse spermatozoa. The objectives of this study were to determine the osmotic tolerance limits for (1) motility, (2) acrosome integrity, and (3) membrane integrity of mouse spermatozoa from multiple genetic backgrounds including: C57BL/6, BALB/c, FVB, C3H, 129/SVS2 hsd B6C3F1, CB6F1, and ICR. The maintenance of acrosomal and plasma membrane integrity was not affected by genetic background (p=0.13), however, there was an interaction between genetic background and osmolality. In addition, acrosome and plasma membrane integrity was highly correlated within each strain (p<0.01). In contrast to acrosome and plasma membrane integrity, the motility of spermatozoa from different genetic backgrounds fell sharply on both sides of isosmolality, both with and without return to isosmotic conditions. Exposure to hyposmotic conditions caused morphological changes in the spermatozoa, which inhibited motility. However, this morphological change was not reversible in all cases when returned to isosmotic conditions. The ability to maintain motility in an anisosmotic media was affected by genetic background, osmolality as well as the interaction between genetic background and osmolality (p<0.05). In conclusion, mice with different genetic backgrounds appear to have similar tolerance to osmotic changes in terms of sperm acrosome and plasma membrane integrity; however, the ability to maintain motility differs between genetic backgrounds.

Acrosome↗
PubMed1986

Specific integral dose: a reconsideration of the integral dose concept.

The integral dose represents the total energy deposited by ionizing radiation within a body. Its distribution within normal tissues can be quite variable, depending upon beam energy and radiation technique. A close look at the concept of integral dose in its various forms should be helpful in quantifying the radiation burden borne by healthy tissues outside of the target volume. The integral dose, integral target dose and relative integral target dose will be reviewed, and the concept of specific integral dose will be introduced, using as examples the external irradiation of the urinary bladder employing various energies and techniques, as well as the intracavitary therapy of gynecological cancer using an afterloading apparatus.

Brachytherapy↗
PubMed1991

Activities of human immunodeficiency virus (HIV) integration protein in vitro: specific cleavage and integration of HIV DNA.

Growth of human immunodeficiency virus (HIV) after infection requires the integration of a DNA copy of the viral RNA genome into a chromosome of the host. Here we present a simple in vitro system that carries out the integration reaction and the use of this system to probe the mechanism of integration. The only HIV protein necessary is the integration (IN) protein, which has been overexpressed in insect cells and then partially purified. DNA substrates are supplied as oligonucleotides that match the termini of the linear DNA product of reverse transcription. In the presence of HIV IN protein, oligonucleotide substrates are cleaved to generate the recessed 3' ends that are the precursor for integration, and the cleaved molecules are efficiently inserted into a DNA target. Analysis of reaction products reveals that HIV IN protein joins 3' ends of the viral DNA to 5' ends of cuts made by IN protein in the DNA target. We have also used this assay to characterize the sequences at the ends of the viral DNA involved in integration. The assay provides a simple screen for testing candidate inhibitors of HIV IN protein; some such inhibitors might have useful antiviral activity.

Base Sequence↗
PubMed2001

HIV-1 preintegration complexes preferentially integrate into longer target DNA molecules in solution as detected by a sensitive, polymerase chain reaction-based integration assay.

After entering a cell and undergoing reverse transcription, the retroviral genome is contained in a preintegration complex (PIC) that mediates its integration into host cell DNA. PICs have been shown to prefer torsionally strained DNA, but the effect of target DNA length has not been previously examined. In this report, concatemerization of a repeating 105-base pair unit was used to vary target DNA length independently from basic DNA sequence, while maintaining both PICs and target DNAs in solution. Integration junctions were quantified by real-time fluorescence-monitored polymerase chain reaction amplification using primers in the viral long terminal repeat and the target DNA. Unreacted target DNA severely inhibited the post-reaction polymerase chain reaction detection step, requiring its removal using lambda exonuclease digestion. Integration into a 32-unit concatemer of target DNA was markedly more efficient than integration into a monomeric unit, indicating that longer target DNA was preferred. This substrate was used to construct a simple, robust, and adaptable assay that can serve as a method for studying the host cell factors that enhance PIC integration, and as a drug discovery platform for integration inhibitors active against PICs.

Binding, Competitive↗
PubMed2005

Reduced community integration in persons following traumatic brain injury, as measured on the Community Integration Measure: an exploratory analysis.

PURPOSE: To explore the community integration of individuals who had suffered a Traumatic Brain Injury (TBI) and compare this to members of the general public. METHOD: An independent groups design explored differences in three groups' levels of community integration. These groups consisted of ten survivors of TBI, ten male and ten female controls and were measured using The Community Integration Measure (CIM). All participants were resident in Northern Ireland (NI). The brain injured participants were drawn from a Belfast-based social skills programme. RESULTS: Mann-Whitney U tests showed a statistically significant difference between female controls and brain-injured individuals (U = 26.50, N(1) = 10, N(2) = 10, p = 0.037, one-tailed). CONCLUSION: Females were more integrated into their communities than males, who were, in turn, more integrated than brain injured individuals. It would appear that brain injury survivors are doubly disadvantaged. Their gender (mainly male), and the injury itself, conspire to reduce their integration within the wider community.

Adult↗