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Analysis of secondary integration sites for IS117 in Streptomyces lividans and their role in the generation of chromosomal deletions.

IS117, the 2.6 kb mini-circle of Streptomyces coelicolor A3(2), is a transposable element previously shown to be integrated into two distant sites in the chromosome. When introduced into S. lividans, IS117 integrates into one preferred chromosomal site, but when this site was artificially deleted, IS117 integrated into many secondary sites. Nucleotide sequence analysis of several secondary integration sites revealed varying degrees of similarity with the preferred site, but no consensus sequence. Nevertheless, sites more similar to the preferred site tended to be occupied more often than those that are less similar. Insertion of IS117 into secondary sites in the chromosome of S. lividans sometimes mediated chromosomal rearrangements. It was shown that some strains containing IS117 integrated into secondary sites had suffered deletions of chromosomal DNA. Deletions were adjacent to the inserted element and were at least several kilobases long. The proposed model implicates homologous recombination between IS117 copies integrated into two different secondary sites in the same chromosome as a cause of the deletions.

Base Sequence↗

Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe.

In order to elucidate the mechanisms of illegitimate recombination in eukaryotes, we have studied the structure of DNA fragments integrated by illegitimate recombination into the genome of fission yeast. Nonhomologous recombination was rarely identified when a long region of homology with the chromosomal leu1+ gene was present in the introduced leu1::ura4+ DNA fragment; but a decrease in length of homology leads to an increase in the ratio of non-homologous to homologous recombination events. The introduced DNA fragments were integrated into different sites in the chromosomes by nonhomologous recombination. The results suggested that there are multiple modes of integration; most events simply involve both ends of the fragments, while in other cases, fragments were integrated in a more complicated manner, probably via circularization or multimerization. To analyze the mechanism of the major type of integration, DNA fragments containing the recombination junctions of three recombinants were amplified by inverted polymerase chain reaction (IPCR) and their nucleotide sequences were determined. There was no obvious homology between introduced DNA and chromosomal DNA at these recombination sites. Furthermore it was found that each terminal region of the introduced DNA was deleted, but that there were no or very small deletions in the target sites of chromosomal DNA. Two models are proposed to explain the mechanism of nonhomologous integration.

Base Sequence↗

Replacement-like recombination induced by an integration vector with a murine homology flank at the immunoglobulin heavy-chain locus in mouse and rat hybridoma cells.

Vectors for homologous recombination are commonly designed as replacement or integration constructs. We have evaluated integration vectors for the substitution of the immunoglobulin heavy-chain constant region by various human isotypes in mouse and rat hybridomas. It is known that under certain circumstances replacement vectors exhibit a lower target efficiency and can be incorporated by integration events. Conversely, we show here that an integration vector can undergo a replacement event despite having free homologous adjacent DNA ends, which would be expected to initiate integration according to the double-strand break repair model. Moreover, in cases of replacement recombination the 5' crossover is not necessarily located within the homology region, thereby giving rise to a truncated gene product. Whether or not the replacement leads to such deletions is clearly dependent on the isotypes involved in the targeting reaction. The fact that the vector is correctly targeted to the heavy-chain locus, but that the homology region is not always the site of recombination, points to a novel recombination mechanism that may be specific for the immunoglobulin loci and that seems to be predominant even in the presence of the free homologous adjacent ends of an integration vector. Furthermore we demonstrate that homologous recombination at the heavy-chain locus is also possible between sequences from different species. The implications of our findings for the production of chimeric antibodies are discussed.

Animals↗

A proposed neural network for the integrator of the oculomotor system.

Single-unit recordings, stimulation studies, and eye movement measurements all indicate that the firing patterns of many oculomotor neurons in the brain stem encode eye-velocity commands in premotor circuits while the firing patterns of extraocular motoneurons contain both eye-velocity and eye-position components. It is necessary to propose that the eye-position component is generated from the eye-velocity signal by a leaky hold element or temporal integrator. Prior models of this integrator suffer from two important problems. Since cells appear to have a steady, background signal when eye position and velocity are zero, how does the integrator avoid integrating this background rate? Most models employ some form of lumped, positive feedback the gain of which must be kept within totally unreasonable limits for proper operation. We propose a lateral inhibitory network of homogeneous neurons as a model for the neural integrator that solves both problems. Parameter sensitivity studies and lesion simulations are presented to demonstrate robustness of the model with respect to both the choice of parameter values and the consequences of pathological changes in a portion of the neural integrator pool.

Animals↗

Site-specific integration and excision of pMEA100 in Nocardia mediterranei.

Nocardia mediterranei strain LBG A3136 contains the 23.7 kb element pMEA100 in a chromosomally integrated form as well as in the free state (Moretti et al. 1985). The integrated form of this element can be excised precisely from the Nocardia chromosome without any accompanying rearrangements in flanking chromosomal DNA. After transfer into plasmid-free mutant strains, pMEA100 reintegrates site specifically into its original chromosomal locus. The exact mapping of the pMEA100 integration site was accomplished by restriction analysis and DNA sequencing. The attachment site of pMEA100, the junctions of its integrated form and plasmid-free chromosomal DNA of N. mediterranei contain an identical 47 bp long sequence which is probably required for site-specific recombination connected with integration and excision of pMEA100. Only one such sequence was found in the chromosome of pMEA100-free N. mediterranei derivatives as suggested by the single integration locus.

Base Sequence↗

Integration of vector-containing Bacillus subtilis chromosomal DNA by a Campbell-like mechanism.

Using plasmid pHV60, which contains a chloramphenicol resistance (Cmr) gene that is expressed in Bacillus subtilis, a set of transformation-deficient strains of B. subtilis was isolated by insertional mutagenesis. When chromosomal DNA from these mutants was used to transform a transformation-proficient B. subtilis strain, almost all of the Cmr transformants had the mutant phenotype as expected. However, with a frequency of approximately 3 X 10(-4) atypical transformants with the wild-type phenotype were produced. Data concerning amplification of the DNA containing the Cmr marker and duplication of DNA sequences are presented that suggest that these atypical transformants are the result of a Campbell-like integration of the chromosomal DNA containing the integrated plasmid. Transductional mapping showed that in the atypical transformants the vector-containing DNA had a strong tendency to integrate at sites adjacent to the original site of integration, although integration at sites elsewhere on the chromosome was also observed. The production of atypical transformants is explained on the basis of integration of chromosomal DNA by a Campbell-like mechanism. Circularization of vector-containing chromosomal DNA is thought to occur through joining of the extremities of single-stranded DNA molecules by fortuitous base pairing with an independently entered single-stranded DNA molecule.

Bacillus subtilis↗

An improved neural-network model for the neural integrator of the oculomotor system: more realistic neuron behavior.

The discharge rates of premotor, brain-stem neurons that create eye movements modulate in relation to eye velocity yet firing rates of extraocular motoneurons contain both eye-position and eye-velocity signals. The eye-position signal is derived from the eye-velocity command by means of a neural network which functions as a temporal integrator. We have previously proposed a network of lateral-inhibitory neurons that is capable of performing the required integration. That analysis centered on the temporal aspects of the signal processing for a limited class of idealized inputs. All of its cells were identical and carried only the integrated signal. Recordings in the brain stem, however, show that neurons in the region of the neural integrator have a variety of background firing rates, all carry some eye-velocity signal as well as the eye-position signal, and carry the former with different strengths depending on the type of eye movement being made. It was necessary to see if the proposed model could be modified to make its neurons more realistic. By modifying the spatial distribution of afferents to the network, we demonstrate that the same basic model functions properly in spite of afferents with nonuniform background firing rates. To introduce the eye-velocity signal a double-layer network, consisting of inhibitory and excitatory cells, was necessary. By presenting the velocity input to only local regions of this network it was shown that all cells in the network still carried the integrated signal and that its cells could carry different eye-velocity signals for different types of eye movements. Thus, this model stimulates quantitatively and qualitatively, the behavior of neurons seen in the region of the neural integrator.

Brain Stem↗

An integrated blood volume pulse biofeedback system for migraine treatment.

A blood volume pulse (BVP) biofeedback system is described that integrates BVP amplitude to provide a signal appropriate for auditory feedback. In comparison to binary BVP feedback methods, this integrated system offers the advantages of continuous feedback and increased scoring ease. The validity of this system was established by correlating the integrated BVP output with trough-to-peak measurements of the raw BVP signal during unassisted relaxation and temporal BVP biofeedback with eight migraine headache patients. Within-subject correlations of the integrated and raw BVP outputs ranged from .82 to .98 (means = .95). Although the integrated method admits unwanted BVP changes in rate, correlation analyses showed this confound factor to be small. Increments in biofeedback training effects were observed during the treatment course. Substantive migraine relief was achieved by the end of treatment and therapeutic gains were maintained at 1-year follow-up. In conclusion, it appears that this method successfully presents continuous auditory feedback from an integrated BVP signal resulting in therapeutic benefits to migraineurs.

Adolescent↗

Integration of bovine leukaemia virus at all stages of enzootic bovine leukosis.

Integration of bovine leukaemia virus DNA was investigated at all stages of infection in cattle. We report here the detection of integrated proviral DNA in the majority of antibody positive animals without lymphocytosis. In all but one case virus was integrated at a number of different sites. Hybridization analysis failed to detect proviral sequences in animals shown to be BLV antibody-negative by the Agar Gel Immunodiffusion assay. The pattern of integration in leukocytes from animals with persistent lymphocytosis was similar to that in sero-positive animals without lymphocytosis in that multiple sites of integration were evident. As reported by others only one or a few sites of integration were detected in tumours from enzootic bovine leukosis animals. Tumours from different sites in individual animals were either monoclonal or oligoclonal.

Animals↗

A model for the mechanism of precise integration of a microinjected transgene.

A unique transgenic mouse line has undergone transgene integration in a very precise fashion. The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed. In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined. A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene. The cellular sequences at the transgene 5' and 3' joins are contiguous in the wild type. The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer. Analysis of the site of insertion has revealed a 5 bp homology between the 5' end of the transgene and the cellular sequences. In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites of DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, the Escherichia coli Chi site and the meiotic recombination hotspot within the E beta gene of the murine major histocompatibility complex. This clustering of features is likely to have been factorial in the integrity of the insertion event. A model depicting the mechanism of this precise integration is proposed.

Animals↗

Integrated structures of HBV DNA in chronic hepatitis and hepatoma tissues.

Cellular DNAs of chronically hepatitis B virus (HBV)-infected human livers were analysed by Southern blot hybridization for the presence of integrated HBV DNA. In 15 out of 16 tissue samples, random HBV DNA integration was evident. By molecular cloning and structural analyses of 19 integrants from 3 chronic hepatitis tissues, rearrangement of HBV DNA with inverted duplication or translocation of cellular flanking DNA at the virus-cell junction was noted. Thus, the rearrangement of HBV DNA or cellular flanking DNA not to be a specific incident of HCC formation. Analyses of various integrants bearing HBV DNA rearrangement and their cellular counterpart DNAs failed to indicate any gross structural alteration in cellular DNA except for a small deletion at the integration sites, indicating HBV DNA rearrangement with inverted duplication to possibly occur prior to integration. Based on nucleotide sequencing analyses of virus-virus junctions, a mechanism of this inverted duplication of HBV DNA is proposed, in which an illegitimate recombination may take place by means of a patchy homology on one side of adjoining viral sequences.

Base Sequence↗

The stimulus integration area for horizontal vergence.

Over what region of space are horizontal disparities integrated to form the stimulus for vergence? The vergence system might be expected to respond to disparities within a small area of interest to bring them into the range of precise stereoscopic processing. However, the literature suggests that disparities are integrated over a fairly large parafoveal area. We report the results of six experiments designed to explore the spatial characteristics of the stimulus for vergence. Binocular eye movements were recorded using magnetic search coils. Each dichoptic display consisted of a central target stimulus that the subject attempted to fuse, and a competing stimulus with conflicting disparity. In some conditions the target was stationary, providing a fixation stimulus. In other conditions, the disparity of the target changed to provide a vergence-tracking stimulus. The target and competing stimulus were combined in a variety of conditions including those in which (1) a transparent textured-disc target was superimposed on a competing textured background, (2) a textured-disc target filled the centre of a competing annular background, and (3) a small target was presented within the centre of a competing annular background of various inner diameters. In some conditions the target and competing stimulus were separated in stereoscopic depth. The results are consistent with a disparity integration area with a diameter of about 5 degrees. Stimuli beyond this integration area can drive vergence in their own right, but they do not appear to be summed or averaged with a central stimulus to form a combined disparity signal. A competing stimulus had less effect on vergence when separated from the target by a disparity pedestal. As a result, we propose that it may be more useful to think in terms of an integration volume for vergence rather than a two-dimensional retinal integration area.

Adult↗

Travel distance estimation from visual motion by leaky path integration.

Visual motion can be a cue to travel distance when the motion signals are integrated. Distance estimates from visually simulated self-motion are imprecise, however. Previous work in our labs has given conflicting results on the imprecision: experiments by Frenz and Lappe had suggested a general underestimation of travel distance, while results from Redlick, Jenkin and Harris had shown an overestimation of travel distance. Here we describe a collaborative study that resolves the conflict by tracing it to differences in the tasks given to the subjects. With an identical set of subjects and identical visual motion simulation we show that underestimation of travel distance occurs when the task involves a judgment of distance from the starting position, and that overestimation of travel distance occurs when the task requires a judgment of the remaining distance to a particular target position. We present a leaky integrator model that explains both effects with a single mechanism. In this leaky integrator model we introduce the idea that, depending on the task, either the distance from start, or the distance to target is used as a state variable. The state variable is updated during the movement by integration over the space covered by the movement, rather than over time. In this model, travel distance mis-estimation occurs because the integration leaks and because the transformation of visual motion to travel distance involves a gain factor. Mis-estimates in both tasks can be explained with the same leak rate and gain in both conditions. Our results thus suggest that observers do not simply integrate traveled distance and then relate it to the task. Instead, the internally represented variable is either distance from the origin or distance to the goal, whichever is relevant.

Adult↗

Sequential cloned gene integration in the yeast Kluyveromyces lactis.

Two integrating vectors developed for use in Saccharomyces cerevisiae were successfully employed for cloned gene integration in the yeast Kluyveromyces lactis. A delta-integrating vector carrying the dominant selection marker neo allowed tandem integrations of a CUP1p-lacZ cassette into one or two chromosomal sites. A delta/UB-integrating vector, which contains a reusable selection cassette, enabled multiple rounds of integration and the sequential insertion of stable, dispersed copies of CUP1p-lacZ. Subsequent gene expression was closely correlated with integrated copy number illustrating the promise of this method for metabolic engineering in K. lactis. While both vectors contain an S. cerevisiae delta target sequence, the presence of delta-like elements in K. lactis has not been confirmed. Given the degree of illegitimate recombination in this yeast species, the insertions likely occurred at random locations in the chromosomes.

Chromosomes, Fungal↗

Construction of a food-grade multiple-copy integration system for Lactococcus lactis.

A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori+) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori+ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-crossover integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5-15) x 10(-2) copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest.

Blotting, Southern↗

Integrating social preference in GIS-aided planning for forestry and conservation activities: a case study from rural SE Asia.

Land-use planning using geographic information systems (GIS) commonly emphasizes biophysical spatial data; however planning can be improved by integrating spatial sets of socioeconomic data into the GIS. As an example, we compared a traditional GIS-aided forestry planning protocol that considered only biophysical suitability, with an integrated GIS-aided approach that incorporated both biophysical and socioeconomic suitability. The analyses were conducted for the planning of plantation investments in the Kyaukpadaung Township in the dry zone of central Myanmar. The traditional approach used three biophysical layers for suitability: land use, slope, and accessibility. In contrast, the integrated GIS approach included biophysical suitability data, perceptions and preferences of local villagers towards forestry (social suitability), and quantitative socioeconomic data. The results indicated that the integrated approach provided two principal benefits over the traditional method. First, the integrated method resulted in a more precise idea of suitable sites for plantation investment that could benefit more rural people and also lead to greater investment efficiency. Second, incorporating social preference into the GIS takes into account the crucial element of social capital (viz., social preference), which should lead to higher levels of community acceptance of plantation projects because those plantations would be established on socially suitable land. A second GIS exercise showed how conservation investment decisions could be informed using the integrated method. The results of this study support the idea that GIS-aided planning activities can be enhanced through the incorporation of social data into the analysis. When applicable, spatial data collection efforts for GIS-based planning exercises should incorporate spatial socioeconomic data.

Asia, Southeastern↗

Towards an integrated approach to natural hazards risk assessment using GIS: with reference to bushfires.

This paper develops a GIS-based integrated approach to risk assessment in natural hazards, with reference to bushfires. The challenges for undertaking this approach have three components: data integration, risk assessment tasks, and risk decision-making. First, data integration in GIS is a fundamental step for subsequent risk assessment tasks and risk decision-making. A series of spatial data integration issues within GIS such as geographical scales and data models are addressed. Particularly, the integration of both physical environmental data and socioeconomic data is examined with an example linking remotely sensed data and areal census data in GIS. Second, specific risk assessment tasks, such as hazard behavior simulation and vulnerability assessment, should be undertaken in order to understand complex hazard risks and provide support for risk decision-making. For risk assessment tasks involving heterogeneous data sources, the selection of spatial analysis units is important. Third, risk decision-making concerns spatial preferences and/or patterns, and a multicriteria evaluation (MCE)-GIS typology for risk decision-making is presented that incorporates three perspectives: spatial data types, data models, and methods development. Both conventional MCE methods and artificial intelligence-based methods with GIS are identified to facilitate spatial risk decision-making in a rational and interpretable way. Finally, the paper concludes that the integrated approach can be used to assist risk management of natural hazards, in theory and in practice.

Conservation of Natural Resources↗

Influence of homology size and polymorphism on plasmid integration in the yeast CYC1 DNA region.

We studied the influence of homology size and polymorphism on the integration of circular plasmids into the yeast CYC1 region. The plasmids used also contained the URA3 gene, and the proportion of Ura+ transformants resulting from plasmid integration into the CYC1 region was determined by Southern-blot analysis. A size-dependent decrease in integration into the CYC1 region was observed from 858 bp to 363 bp of homology. However, with a homology size of 321, 259 or 107 bp, about 2% of the transformants still contained plasmid molecules integrated in the CYC1 region. A single point mutation in the 858-bp fragment decreased the proportion of integrations to the CYC1 gene, but the presence of additional mutations did not have a cumulative effect. For plasmids isolated in a single-stranded (ss) form, the presence of two or six point mutations did not influence integration. These results were compared with those obtained in other assays designed to study substrate requirements for homologous recombination.

Cytochrome c Group↗