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PubMed2001

Identification of genes required for excision of CTnDOT, a Bacteroides conjugative transposon.

Integrated self-transmissible elements called conjugative transposons have been found in many different bacteria, but little is known about how they excise from the chromosome to form the circular intermediate, which is then transferred by conjugation. We have now identified a gene, exc, which is required for the excision of the Bacteroides conjugative transposon, CTnDOT. The int gene of CTnDOT is a member of the lambda integrase family of recombinases, a family that also contains the integrase of the Gram-positive conjugative transposon Tn916. The exc gene was located 15 kbp from the int gene, which is located at one end of the 65 kbp element. The exc gene, together with the regulatory genes, rteA, rteB and rteC, were necessary to excise a miniature form of CTnDOT that contained only the ends of the element and the int gene. Another open reading frame (ORF) in the same operon and upstream of exc, orf3, was not essential for excision and had no significant amino acid sequence similarity to any proteins in the databases. The deduced amino acid sequence of the CTnDOT Exc protein has significant similarity to topoisomerases. A small ORF (orf2) that could encode a small, basic protein comparable with lambda and Tn916 excision proteins (Xis) was located immediately downstream of the CTnDOT int gene. Although Xis proteins are required for excision of lambda and Tn916, orf2 had no effect on excision of the element. Excision of the CTnDOT mini-element was not affected by the site in which it was integrated, another difference from Tn916. Our results demonstrate that the Bacteroides CTnDOT excision system is tightly regulated and appears to be different from that of any other known integrated transmissible element, including those of some Bacteroides mobilizable transposons that are mobilized by CTnDOT.

Amino Acid Sequence↗

PubMed1998

Multiple control elements are required for expression of the human CD34 gene.

Two cis regulatory elements of the human CD34 gene, the promoter and a 3' enhancer, have previously been described. In transient transfection assays, the promoter was not sufficient to direct cell type specific expression. In contrast, the 3' enhancer was active only in CD34+ cell lines, suggesting that this element might be responsible for stem cell-restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive sites, we detected a strong CD34+ cell type-specific site in intron 4. This region, however, did not work as an enhancer by itself. By analyzing stable transfectants and transgenic animals, we demonstrated that the 3' enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression.

Animals↗

PubMed1999

Contour integration and scale combination processes in visual edge detection.

Contours in the natural visual environment consist mainly of edges which are spatially broad-band and whose (cosinusoidal) components have arrival phases close to +/-90 deg. Because early visual processing is thought to be based on a local Fourier description, the representation of edges requires two forms of filter combination: scale integration (filter combination across spatial frequency) and contour integration (filter combination across space). In order to determine how these two types of combination fit together, we determined spatial-frequency tuning for the detection of contours composed of broadband edge elements, alternating with narrow-band Gabor elements. A contour integration system operating independently at a number of spatial scales should be able to ignore the distracting influence of edge structure in such patterns. However, subjects cannot ignore edge structure indicating that local phase-alignment across spatial scale is coded prior to, or concurrent with, contour integration. Moreover, unlike contours composed of Gabors, the bandwidth of local elements is important for edge integration; the coding of element bandwidth seems to be dependent on the phase alignment of features across spatial frequency.

Computer Simulation↗

PubMed1994

Integration of a vector containing a repetitive LINE-1 element in the human genome.

Mammalian cells contain numerous nonallelic repeated sequences, such as multicopy genes, gene families, and repeated elements. One common feature of nonallelic repeated sequences is that they are homeologous (not perfectly identical). Our laboratory has been studying recombination between homeologous sequences by using LINE-1 (L1) elements as substrates. We showed previously that an exogenous L1 element could readily acquire endogenous L1 sequences by nonreciprocal homologous recombination. In the study presented here, we have investigated the propensity of exogenous L1 elements to be involved in a reciprocal process, namely, crossing-overs. This would result in the integration of the exogenous L1 element into an endogenous L1 element. Of over 400 distinct integration events analyzed, only 2% involved homologous recombination between exogenous and endogenous L1 elements. These homologous recombination events were imprecise, with the integrated vector being flanked by one homologous and one illegitimate junction. This type of structure is not consistent with classical crossing-overs that would result in two homologous junctions but rather is consistent with one-sided homologous recombination followed by illegitimate integration. Contrary to what has been found for reciprocal homologous integration, the degree of homology between the exogenous and endogenous L1 elements did not seem to play an important role in the choice of recombination partners. These results suggest that although exogenous and endogenous L1 elements are capable of homologous recombination, this seldom leads to crossing-overs. This observation could have implications for the stability of mammalian genomes.

Cell Line↗

PubMed2000

Dendritic integration of excitatory synaptic input.

A fundamental function of nerve cells is the transformation of incoming synaptic information into specific patterns of action potential output. An important component of this transformation is synaptic integration--the combination of voltage deflections produced by a myriad of synaptic inputs into a singular change in membrane potential. There are three basic elements involved in integration: the amplitude of the unitary postsynaptic potential; the manner in which non-simultaneous unitary events add in time (temporal summation), and the addition of unitary events occurring simultaneously in separate regions of the dendritic arbor (spatial summation). This review discusses how passive and active dendritic properties, and the functional characteristics of the synapse, shape these three elements of synaptic integration.

Action Potentials↗

PubMed2003

Characterization of two alternative promoters for integrase expression in the clc genomic island of Pseudomonas sp. strain B13.

The clc genomic island is a 105 kb integrative and conjugative element (ICE) in Pseudomonas sp. strain B13, which encodes metabolism of 3-chlorocatechol. The clc island is integrated in a tRNAGly gene, but can excise and form a circular intermediate in which both ends are connected. The integrase gene (intB13) of the clc genomic island is located at the right end, 202 bp from the junction site facing inwards. Fragments upstream of intB13 in the circular form and in the integrated form were fused to a promoterless gfp gene for Green Fluorescent Protein and introduced in monocopy onto the chromosome of strain B13. Quantitative GFP fluorescence measurements in individual cells of the different B13-derivatives revealed that the circular form fragment contained a strong constitutive promoter (Pcirc) driving intB13 expression in all cells. By using primer extension Pcirc could be mapped near the left end of the clc element and Pcirc can therefore only control intB13 expression when left and right ends are connected as in the circular form. Expression from intB13 upstream fragments from the integrated clc element was weaker than that from Pcirc and only occurred in maximally 15% of individual cells in a culture. A promoter (Pint) could be roughly mapped in this region by using reverse-transcription PCR and by successively shortening the fragment from the 5' end. Transposon mutants in cloned left end sequences of the clc element were selected which had lost the activation potential on the Pint promoter and those which resulted in overexpression of GFP from Pint. The DNA sequence of the region of the transposon insertions pointed to a relatively well conserved area among various other genomic islands. The activator mutants mapped in an open reading frame (ORF) encoding a 175 amino acid protein without any significant similarity to functionally characterized proteins in the databases.

Base Sequence↗

PubMed1998

In vivo determination of homogenised mechanical characteristics of human tibia: application to the study of tibial torsion in vivo.

OBJECTIVE: The study presents a method allowing the in vivo homogenised characteristics of the tibiae of children to be assessed. DESIGN: Studies have been performed on two groups of children: six normal children, aged from 5 to 16 yr, and on four children, aged from 8 to 11 yr with tibial deformities. We analysed the tibial transverse sections from CT scans performed on the left tibia of each child. BACKGROUND: Most tibial torsion studies have only been based on geometrical parameters. Our study integrated mechanical and geometrical considerations. METHODS: The finite element models and integration of mechanical properties were performed from CT scans. Then homogenised mechanical characteristics (tensile stiffness, flexural stiffness and torsional stiffness) were calculated. RESULTS: The homogenised mechanical characteristics decrease between 20 to 80% of the tibial length. The values increased with age for both groups of children. Children with abnormalities seem to have values of tibial rigidities comparable with those of normal tibiae. CONCLUSIONS: By considering the mechanical and geometrical properties of the tibia in our study, we showed that the bone stiffness of children is not altered with torsional deformities. RELEVANCE: Torsional tibial abnormalities of children are a frequent phenomenon which may have important consequences on gait and joints. The method developed could be used as an objective assessment of bone rigidities for analysing tibial disorders such as torsional abnormalities of varying severity.

Journal Article↗

PubMed1998

Understanding the costs of ambulatory care training.

While patient care has been shifting to the ambulatory setting, the education of health care professionals has remained essentially hospital-based. One factor discouraging the movement of training into community-based ambulatory settings is the lack of understanding of what the costs of such training are and how these costs might be offset. The authors describe a model for ambulatory care training that makes it easier to generalize about to quantify its educational costs. Since ambulatory care training does not exist in a vacuum separate from inpatient education, the model is compatible with the way hospital-based education costs are derived. Thus, the model's elements can be integrated with comparable hospital-based training cost elements in a straightforward way to allow a total-costing approach. The model is built around two major sets of variables affecting cost. The first comprises three types of costs--direct, indirect, and infrastructure--and the second consists of factors related to the training site and factors related to the educational activities of the training. The model is constructed to show the various major ways these two sets of variables can influence training costs. With direct Medicare funding for some ambulatory-setting-based education pending, and with other regulatory and market dynamics already in play, it is important that educators, managers, and policymakers understand how costs, the characteristics of the training, and the characteristics of the setting interact. This model should assist them. Without generalizable cost estimates, realistic reimbursement policies and financial incentives cannot be formulated, either in the broad public policy context or in simple direct negotiations between sites and sponsors.

Ambulatory Care↗

PubMed2005

The repA gene of the linear Yersinia enterocolitica prophage PY54 functions as a circular minimal replicon in Escherichia coli.

The Yersinia enterocolitica prophage PY54 replicates as a linear DNA molecule with covalently closed ends. For replication of a circular PY54 minimal replicon that has been derived from a linear minireplicon, two phage-encoded loci are essential in Escherichia coli: (i) the reading frame of the replication initiation gene repA and (ii) its 212-bp origin located within the 3' portion of repA. The RepA protein acts in trans on the origin since we have physically separated the PY54 origin and repA onto a two-plasmid origin test system. For this trans action, the repA 3' end carrying the origin is dispensable. Mutagenesis by alanine scan demonstrated that the motifs for primase and for nucleotide binding present in the protein are essential for RepA activity. The replication initiation functions of RepA are replicon specific. The replication initiation proteins DnaA, DnaG, and DnaB of the host are unable to promote origin replication in the presence of mutant RepA proteins that carry single residue exchanges in these motifs. The proposed origins of the known related hairpin prophages PY54, N15, and PKO2 are all located toward the 3' end of the corresponding repA genes, where several structure elements are conserved. Origin function depends on the integrity of these elements.

3' Untranslated Regions↗

PubMed2000

Integration of foveal orientation signals: distinct local and long-range spatial domains.

Human observers can discriminate the orientation of a stimulus configuration composed of a pair of collinear visual patterns much better than that of a single component pattern alone. Previous investigations of this type of orientation signal integration and of other similar visual integrative functions have shown that, for closely spaced elements, there is integration only for stimuli with the same contrast polarity (i.e., both lighter or both darker than the background) but, at greater separations, integration is independent of contrast polarity. Is this effect specific to differences in contrast polarity, which is known to be an important parameter in the organization of the visual system, or might there be a cluster of other stimulus dimensions that show similar effects, indicating a more widespread distinction between the processes limiting integration at local and long-range spatial scales? Here, we report a similar distance dependence for orientation signal integration across stimulus differences in binocular disparity, direction of motion, and direction of figure-ground assignment. We also demonstrate that the selectivity found at short separations cannot be explained only by "end-cuts," the small borders created at the junction of abutting contrasting patterns. These findings imply the existence of two distinct spatial domains for the integration of foveal orientation information: a local zone in which integration is highly selective for a number of salient stimulus parameters and a long-range domain in which integration is relatively unselective and only requires that patterns be roughly collinear.

Contrast Sensitivity↗

PubMed1999

Recently integrated human Alu repeats: finding needles in the haystack.

Alu elements undergo amplification through retroposition and integration into new locations throughout primate genomes. Over 500,000 Alu elements reside in the human genome, making the identification of newly inserted Alu repeats the genomic equivalent of finding needles in the haystack. Here, we present two complementary methods for rapid detection of newly integrated Alu elements. In the first approach we employ computational biology to mine the human genomic DNA sequence databases in order to identify recently integrated Alu elements. The second method is based on an anchor-PCR technique which we term Allele-Specific Alu PCR (ASAP). In this approach, Alu elements are selectively amplified from anchored DNA generating a display or 'fingerprint' of recently integrated Alu elements. Alu insertion polymorphisms are then detected by comparison of the DNA fingerprints generated from different samples. Here, we explore the utility of these methods by applying them to the identification of members of the smallest previously identified subfamily of Alu repeats in the human genome termed Ya8. This subfamily of Alu repeats is composed of about 50 elements within the human genome. Approximately 50% of the Ya8 Alu family members have inserted in the human genome so recently that they are polymorphic, making them useful markers for the study of human evolution.

Animals↗

PubMed2005

[Digital teaching archive. Concept, implementation, and experiences in a university setting].

Film-based teaching files require a substantial investment in human, logistic, and financial resources. The combination of computer and network technology facilitates the workflow integration of distributing radiologic teaching cases within an institution (intranet) or via the World Wide Web (Internet). A digital teaching file (DTF) should include the following basic functions: image import from different sources and of different formats, editing of imported images, uniform case classification, quality control (peer review), a controlled access of different user groups (in-house and external), and an efficient retrieval strategy. The portable network graphics image format (PNG) is especially suitable for DTFs because of several features: pixel support, 2D-interlacing, gamma correction, and lossless compression. The American College of Radiology (ACR) "Index for Radiological Diagnoses" is hierarchically organized and thus an ideal classification system for a DTF. Computer-based training (CBT) in radiology is described in numerous publications, from supplementing traditional learning methods to certified education via the Internet. Attractiveness of a CBT application can be increased by integration of graphical and interactive elements but makes workflow integration of daily case input more difficult. Our DTF was built with established Internet instruments and integrated into a heterogeneous PACS/RIS environment. It facilitates a quick transfer (DICOM_Send) of selected images at the time of interpretation to the DTF and access to the DTF application at any time anywhere within the university hospital intranet employing a standard web browser. A DTF is a small but important building block in an institutional strategy of knowledge management.

Computer-Assisted Instruction↗

PubMed2007

Mobilization and evolutionary history of miniature inverted-repeat transposable elements (MITEs) in Beta vulgaris L.

We have identified three families of miniature inverted-repeat transposable elements (VulMITEs) in the genome of sugar beet (Beta vulgaris L.), evidently derived from a member of the Vulmar family of mariner transposons. While VulMITEs I are typical stowaway-like MITEs, VulMITEs II and VulMITEs III are rearranged stowaway elements of increased size. The integration of divergent moderately and highly repetitive sequences into VulMITEs II and, in particular in VulMITEs III, respectively, shows that amplification of repetitive DNA by MITEs contribute to the increase of genome size with possible implications for plant genome evolution. Fluorescent in-situ hybridization (FISH), for the first time visualizing stowaway MITE distribution on plant chromosomes, revealed a dispersed localization of VulMITEs along all B. vulgaris chromosomes. Analysis of the flanking sequences identified a dispersed repeat as target site for the integration of the stowaway element VulMITE I. Recent transposition of VulMITE I, which most likely occurred during the domestication of cultivated beets, was concluded from insertional polymorphisms between different B. vulgaris cultivars and species.

Base Sequence↗

PubMed1987

Genetic organization of insertion element IS2 based on a revised nucleotide sequence.

We identified a transposable element resident in the chromosome of Escherichia coli K-12 strain HB101. This is an approx. 4400-bp-long transposon flanked by two copies of insertion sequence (IS) 1 element in direct orientation. One of the IS1 elements was found to be integrated into an IS2 element between IS2 bp 139 and bp 140 with the large moiety of IS2 within the transposon. The sequence of this part of IS2 differs from the published sequence of galOP-308::IS2 at a number of positions. Restriction analysis of the published allele, however, indicated that both alleles may in fact be identical. Since six of the eight differences found alter open reading frames, the revised sequence presents a new outlook for the potential genetic organization of IS2.

Base Composition↗

PubMedDate not supplied

Research environments that promote integrity.

BACKGROUND: The body of empirical knowledge about research integrity and the factors that promote research integrity in nursing research environments remains small. OBJECTIVE: To propose an internal control model as an innovative framework for the design and structure of nursing research environments that promote integrity. METHODS: An internal control model is adapted to illustrate its use for conceptualizing and designing research environments that promote integrity. RESULTS: The internal control model integrates both the organizational elements necessary to promote research integrity and the processes needed to assess research environments. The model provides five interrelated process components within which any number of research integrity variables and processes may be used and studied: internal control environment, risk assessment, internal control activities, monitoring, and information and communication. DISCUSSION: The components of the proposed research integrity internal control model proposed comprise an integrated conceptualization of the processes that provide reasonable assurance that research integrity will be promoted within the nursing research environment. Schools of nursing can use the model to design, implement, and evaluate systems that promote research integrity. The model process components need further exploration to substantiate the use of the model in nursing research environments.

Communication↗

PubMed1990

Network study of integrated biochemical switching system. I: Connection of basic elements.

As a first step to network study, a series of integrated biochemical switching systems (prototype of artificial neuronic device) was assumed based on Rosen's work and on the results of our previous studies. The effects of an excitatory stimulus on the switching properties of the proposed system were examined using computer simulations. The results can be summarized as follows: (i) the number of excited elements in sequentially connected systems is proportionally related to the value of the excitatory stimulus; (ii) when the introduction of the excitatory stimulus is too late, it can no longer be transmitted to any elements; (iii) the excitatory stimulus (signal) is amplified to a certain limit and is attenuated during propagation; (iv) by assuming several excitatory stimuli and varying their frequencies, the so-called long-term potentiation phenomenon can be observed; (v) supposing reversible interactions between two elements, a continuous switching pattern of the output is observed.

Animals↗

PubMed1993

A composite hormone response element mediates the transactivation of the rat oxytocin gene by different classes of nuclear hormone receptors.

Transcription factors of the steroid/thyroid hormone receptor superfamily are mediators of development and regulation of the brain. Previous studies have shown that the hypothalamic oxytocin (OT) gene is a potential target of these receptors, since its promoter is stimulated by estrogens and thyroid hormone. Here it is shown that the rat OT promoter is stimulated (at least 20-fold) by retinoic acid through two distinct regions in the 5'-flanking region. The major retinoic acid response element was located between nucleotides -172 and -148 and a minor one between nucleotides -112 and -77, as concluded from the transactivation of 5'-deletion mutants and binding to promoter elements by the retinoic acid receptor. Since the -172/-148 element also conferred estrogen and thyroid hormone responsiveness, it can be considered a composite hormone response element. This element contains a natural variant of the direct repeat of the half-site AGGTCA with spacing zero (DR-0) as well as a palindrome. Analysis of the core sequences of this element by site-directed mutagenesis showed that each of the three TGACC motifs integral to this element contributes to the multihormone sensitivity, but the contribution of each motif is different for the individual receptors. In neonatal rats, vitamin A deficiency and retinoic acid supplementation did not cause changes in hypothalamic OT mRNA levels and OT peptide levels in the pituitary gland and plasma. Gel-retarded protein-DNA complexes were formed between the composite hormone response element and extracts of the hypothalamic supraoptic and paraventricular nuclei. The composite hormone response element has a unique configuration and integrates responses of multiple members of the steroid/thyroid hormone receptor superfamily.

Animals↗

PubMed1990

Identification of a second retrotransposon-related element in the genome of Physarum polycephalum.

The repetitive fraction of the genome of the eukaryotic slime mould Physarum polycephalum is dominated by Tp1, a family of retrotransposon-like sequences. Tp1 elements are arranged in scrambled clusters probably arising from integration of the element into copies of its own sequence. The present report describes a second sequence family, Tp2, which has been identified within cloned DNA segments of scrambled Tp1 sequences. Like Tp1, the Tp2 element is structurally related to retrotransposons, having long terminal direct repeats and being flanked by an apparent target site duplication, but its relatively short length (1.68 kb) indicates that it is probably incapable of encoding all the functions necessary for its own mobilisation. Analysis of the coding potential of the Tp2 element supports this view, although a striking homology to a nucleic acid binding domain common to many retrotransposons was identified. As with Tp1, putative regulatory signals can be identified in the LTRs of Tp2. Identical arrangements of Tp2 with respect to Tp1 in more than one independently derived clone indicate that non-functional copies of Tp2 may be mobilised as part of a Tp1 transcriptional unit.

Amino Acid Sequence↗