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PubMed2004

Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells.

The Tol2 transposable element of the Japanese medaka fish belongs to the hAT family of transposons including hobo of Drosophila, Ac of maize, and Tam3 of snapdragon. To date, Tol2 is the only natural transposon in vertebrates that has ever been shown to encode a fully functional transposase. It has not been known, however, whether Tol2 can transpose in vertebrates other than fish. We report here transposition of Tol2 in mouse embryonic stem (ES) cells. We constructed a transposon donor plasmid containing a nonautonomous Tol2 element with the neomycin resistance gene and a helper plasmid capable of expressing the transposase and introduced the donor plasmid with various amounts of the helper plasmid by electroporation into mouse ES cells. The number of G418-resistant ES colonies increased as the amount of helper plasmid was increased, in a dose-dependent manner, indicating that the transposase activity elevated the integration efficiency. These G418-resistant ES colonies were cloned and the structure of the junction of the integrated Tol2 element and the genomic DNA was analyzed by inverse PCR. In those clones, Tol2 was surrounded by mouse genomic sequences and an 8-bp direct repeat was created adjacent to both ends of Tol2, indicating that Tol2 was integrated in the genome through transposition. The Tol2 transposon system is thus active in mouse as well as in fish. We propose that it should be used as a genetic tool to develop novel gene transfer, transgenesis, and mutagenesis methods in mammals.

Animals↗
PubMed2003

Comprehensive analysis of two Alu Yd subfamilies.

Alu elements have inserted in the human genome throughout primate evolution. A small number of Alu insertions have occurred after the divergence of humans from nonhuman primates and therefore should not be present in nonhuman primate genomes. Most of these recently integrated Alu elements are contained with a series of discrete Alu subfamilies that are related to each other based upon diagnostic nucleotide substitutions. We have extracted members of the Alu Yd subfamily that are derivatives of the Alu Y subfamily that share a common 12-bp deletion that defines the Yd lineage from the draft sequence of the human genome. Analysis of the Yd Alu elements resulted in the recovery of two new Alu subfamilies, Yd3 and Yd6, which contain a total of 295 members (198 Yd3 and 97 Yd6). DNA sequence analysis of each of the Alu Yd subfamilies yielded age estimates of 8.02 and 1.20 million years old for the Alu Yd3 and Yd6 subfamilies, respectively. Two hundred Alu Yd3 and Yd6 loci were screened using polymerase chain reaction (PCR) assays to determine their phylogenetic origin and associated levels of human genomic diversity. The Alu Yd3 subfamily appears to have started amplifying relatively early in primate evolution and continued propagating albeit at a low level as many of its members are found in a variety of hominoid (humans, greater and lesser ape) genomes. Only two of the elements are polymorphic in the human genome and absent from the genomes of nonhuman primates. By contrast all of the members of the Alu Yd6 subfamily are restricted to the human genome, with 12% of the elements representing insertion polymorphisms in human populations. A single Alu Yd6 locus contained an independent parallel forward insertion of a paralogous Alu Sq sequence in the owl monkey. These Alu subfamilies are a source of genomic fossil relics for the study of primate phylogenetics and human population genetics.

Alu Elements↗
PubMed1986

Transposition of the maize controlling element "Activator" in tobacco.

Transposition of the maize autonomous controlling element Activator (Ac) and a nonautonomous derivative, Dissociation (Ds), was investigated in tobacco cells. Tobacco protoplasts were transformed with Ti-plasmid vectors that contained Ac or Ds flanked by short maize wx gene sequences. The structures of the elements and surrounding wx and T-DNA sequences were investigated in nine Ac and five Ds tobacco transformants by digestion with restriction enzymes, Southern blotting, and hybridization using specific probes. In four of the nine Ac transformed lines, Ac had excised from its original position in the T-DNA and inserted at new sites in the tobacco genome. Ds did not excise from its original T-DNA position in any of the transformants examined. Two Ac fragments and cellular flanking sequences were cloned from a line of tobacco in which Ac had transposed. Fragments, comprised of sequences flanking the newly integrated Ac elements, were used as hybridization probes to normal tobacco DNA and to the tobacco DNA from which they were isolated. The Ac copies were integrated into repetitive tobacco DNA sequences. Two tobacco fragments containing empty Wx donor sites were cloned from the DNA of the same Ac transformant and sequenced. Both sequences are among the types of excision products observed to result from Ac-catalyzed excision events in maize. Our results indicate that the maize controlling element Ac is capable of self-catalyzed transposition in tobacco.

Journal Article↗
PubMedDate not supplied

Polymorphism near the rat prolactin gene caused by insertion of an Alu-like element.

Primate Alu and rodent Alu-like elements comprise major families of mammalian small dispersed repetitive DNAs. These elements are repeated more than 10(5) times per haploid genome and are found between known genes, in introns and in satellite DNA. Their dispersion throughout the genome and the presence of directly repeated DNA sequences flanking the elements suggest, but do not prove, that they are capable of transposition. We describe here an allelic variation in the 5'-flanking region of the rat prolactin gene that offers the opportunity to examine the sequences of matching regions of two homologous chromosomes which differ in the presence of an Alu-like repetitive DNA element. Our findings support the hypothesis that these elements are integrated into the genome by generating short direct repeats of host DNA.

Alleles↗
PubMedDate not supplied

[Structural instability of co-integrates formed during interaction of plasmid R57 with pB322 and RP1. Possible role of IS1 element in the degradation of co-integrates].

The conjugative plasmid R57 determines resistance to ampicillin and chloramphenicol. Earlier it was shown that R57 encodes site-specific recA-independent recombinase, which acts in cis and resolves IS1-mediated cointegrates arising in the Escherichia coli recA cells between R57 and pBR322. In the present work the properties of the cointegrates between R57 and pBR322 or RP1 arising in the E. coli rec+ strains were studied. It was found that the cointegrates between R57 and pBR322, obtained by mating of the respective biplasmid donors of E. coli rec+ and the rec+ recipients, lost as a result of deletion a large DNA segment of R57 containing determinant Cmr. The resulting hybrid replicons preserved determinants Apr and Tcr of pBR322 and the R57 conjugative properties and were structurally identical. By using plasmid RP1ts12, which is temperature-sensitive in replication, it was demonstrated that in cells rec+ the cointegrates between R57 and RP1 are extremely unstable. On storage they undergo structural degradation mainly affecting the RP1 replicon. The degradation products of the hydrid complex had lost their RP1 genes but preserved the R57 functional determinants. For elucidation of the observed phenomena the properties of the IS1-mediated cointegrates between pBR322:Tn9 and plasmid pBR3.1--deletion derivative of RP1 were studied. It was found that insertion of IS1 sometimes resulted in formation of unstable cointegrates capable of resolving and loosing determinant Cmr with a high frequency. It was suggested that IS1 encodes the site-specific recombinase responsible for resolution of the IS1-mediated cointegrates and deletion generation. Expression of this recombinase appears to be dependent on structure of the insertion sites. The possible role of IS1 and recombinase encoded by it in resolution and structural instability of the cointegrates between R57 and pBR322 or RP1 is discussed.

Chromosome Mapping↗
PubMed2000

A finite-element model for the mechanical analysis of skeletal muscles.

In the present paper, a finite-element model for simulating muscle mechanics is described. Based on nonlinear continuum mechanics an algorithm is proposed that includes the contractile active and passive properties of skeletal muscle. Stress in the muscle is assumed to result from the superposition of a passive and an active part. The passive properties are described by a hyperelastic constitutive material law whereas the active part depends on the fibre length, shortening velocity and an activation function. The constraint of approximate incompressibility of the muscle element is satisfied as a property of the constitutive equations. Because of the nonlinear behaviour of the material and the highly dynamical performance an incremental procedure including iterative methods is used. The advantage of the model over previous formulations is the possibility to integrate the element into an engineering standard finite-element programme ANSYS using advanced numerical tools. The model allows simulations of muscle recruitment, calculations of stress and strain distributions and predictions of muscle shape. Other possible applications are studies of the muscle architecture, the effect of inertia and impacts. First, simple examples are presented.

Animals↗
PubMed2001

Differential control by IHF and cAMP of two oppositely oriented genes, hpt and gcd, in Escherichia coli: significance of their partially overlapping regulatory elements.

The hpt gene, which encodes hypoxanthine phosphoribosyltransferase, is located next to, but transcribed in the opposite direction to, the gcd gene, which codes for a membrane-bound glucose dehydrogenase, at 3.1 min on the Escherichia coli genome. In their promoter-operator region, putative regulatory elements for integration host factor (IHF) and for the complex comprising 3', 5'-cyclic AMP (cAMP) and its receptor protein (CRP) are present, and they overlap the promoters for hpt and gcd, respectively. The involvement of IHF and cAMP-CRP, as well as the corresponding putative cis-acting elements, in the expression of the two genes was investigated by using lacZ operon fusions. In an adenylate cyclase-deficient strain, addition of cAMP increased the expression of hpt and reduced the expression of gcd. In agreement with this observation, the introduction of mutations into the putative binding element for the cAMP-CRP complex enhanced the expression of gcd. In contrast, mutations introduced into the putative IHF-binding elements increased the level of hpt expression. Similar results were obtained with IHF-defective strains. Thus, the expression of the two genes is regulated in a mutually exclusive manner. Additional experiments with mutations at the -10 sequence of the gcd promoter suggest that the binding of RNA polymerase to the hpt promoter interferes with the interaction of RNA polymerase with the gcd promoter, and vice versa.

Bacterial Proteins↗
PubMed1997

Fundamental strategies for skin care.

The Agency for Health Care Policy and Research (AHCPR) identified the need to develop clinical practice guidelines for the prevention and treatment of pressure ulcers, a segment of wound and skin diagnoses. The skin is the first line of defense in protecting the body from constant changes in the environment. Far too often, the attention needed for the skin is only realized after the integrity has been disrupted. This article examines the anatomy and physiology of the skin, factors that affect the skin (aging, bathing, cleansers, dryness, friction, lotions, moisturizers, nutrition, soaps and shearing forces), indications and contraindications of topical skin care products, and AHCPR's recommended interventions. These elements are integral to clinicians in proactively developing sound skin care prevention strategies. Yet without a thorough understanding of all components, the skin's integrity remains at risk.

Humans↗
PubMed1996

Efficient control of gene expression by single step integration of the tetracycline system in transgenic mice.

Tetracycline-regulated gene expression in eukaryotic cell lines, plants, and transgenic mice has become a powerful tool for the analysis of eukaryotic gene expression and function. The system consists of two plasmids, one encoding the transactivator protein under control of a viral cytomegalovirus promoter, and the second being the tet-operator minimal promoter driving the gene of interest. Here we show that these control elements, when integrated in cis on a single plasmid, allow efficient and tight control of reporter gene expression in vitro and in vivo. Dependent on the route of administration of tetracycline, gene expression can be partially or fully repressed in transgenic mice, whereas removal of the antibiotic induces the reporter gene in various tissues to levels up to 800-fold more than the two-plasmid system. In addition, crossing and analysis of animals transgenic for the individual components of the system are unnecessary, and genetic segregation of the control elements during breeding is prevented.

Animals↗
PubMed1997

Disease management of COPD with pulmonary rehabilitation.

Pulmonary rehabilitation is a set of tools and disciplines that attends to the multiple needs of the COPD patient. It extends beyond standard care by addressing the disabling features of chronic and progressive lung disease. It centers on self-management, exercise, functional training, psychosocial skills, and contributes to the optimization of medical management. Exercise enables other components by building strength, endurance, confidence, and reducing dyspnea. Patients who have undergone rehabilitation often enjoy a reduced need for health-care utilization. On the downside, rehabilitation is a one-time intervention, the benefits of which dissolve over time. The patient's physician is rarely a participant in the program; thus, the physician is at a disadvantage in being able to support a long-term response. Rehabilitation is available to a small percentage of a large patient population who could benefit. Optimal disease management would entail redesigning standard medical care to integrate rehabilitative elements into a system of patient self-management and regular exercise. It should emphasize physician involvement in self-management, which is essential in developing and maintaining an effective exacerbation protocol. Pulmonary rehabilitation should take its place in the mainstream of disease management through its integrative and reconciliative role in the multidisciplinary continuum of services, as defined by the National Institutes of Health, Pulmonary Rehabilitation Research, Workshop of 1994.

Combined Modality Therapy↗
PubMed1993

Sequence relationship of retrotransposable elements R1 and R2 within and between divergent insect species.

R1 and R2 are retrotransposable elements that integrate at specific sites in the 28S ribosomal RNA (rRNA) genes of Bombyx mori and Drosophila melanogaster. We have previously shown that most insect species contain insertions in their 28S genes at the R1 and/or R2 site. We have sequenced the 3' half of R1 and R2 elements from three additional insect species: the fungus gnat, Sciara coprophila (Diptera); the Japanese beetle, Popillia japonica (Colleoptera); and the parasitic wasp, Nasonia vitripennis (Hymenoptera). The elements were obtained by screening lambda phage genomic clones containing rDNA units and by a polymerase chain reaction approach using degenerate primers to conserved sequences in the reverse-transcriptase domain, in combination with a second primer to the 28S gene 3' of the insertion site. Comparisons of the sequences of R1 and R2 from four insect orders suggest that the organization of their open-reading frames has been conserved and is therefore likely to be similar throughout insects. This sequence analysis also indicates that, except for 5' truncations generated during the retrotransposition process itself, most elements have not accumulated mutations that would make them inactive. Popillia japonica and N. vitripennis differed from previously described species, in that (a) P. japonica contained multiple families of R2 and (b) N. vitripennis contained multiple families of R1. Nucleotide sequence identity between these different families is low. Amino acid sequence identity of their open-reading frames averaged only 41% for the R2 families of P. japonica and 35% for the R1 families of N. vitripennis. The presence of multiple highly divergent families of elements within a species suggests either that each insertion family is able to maintain its copy number without eliminating the other families in its competition for a limited number of 28S genes or that there has been extensive horizontal transfer of R1 and R2 elements between insect species.

Amino Acid Sequence↗
PubMed2026

MDV-like endogenous viral elements act as immune rheostats in Aedes cells by modulating defensin A-mediated responses to arboviruses.

Mosquito cell lines are essential tools for arbovirus research. Endogenous viral elements (EVEs) are prevalent in mosquito genomes, yet their functional effects on host immune responses remain unclear, potentially complicating experimental interpretations. In this study, we systematically characterized endogenous mosquito densovirus-like elements (EMLs) within the Aedes aegypti Aag2 cell line and found that these endogenous EMLs are transcriptionally active but translationally defective. The silencing of EML transcripts significantly diminished the replication of Zika virus (ZIKV), Japanese encephalitis virus (JEV), and chikungunya virus (CHIKV), while transiently increasing dengue virus 2 (DENV-2), thereby indicating a virus-dependent regulatory mechanism. Mechanistically, RNA sequencing after EML interference, alongside plasmid-based mimic expression, demonstrated that EML transcripts downregulate defensin A, an antimicrobial peptide produced by mosquitoes. Functional assays using synthetic defensin A showed that this peptide differentially regulates arboviral infection. Binding assays and structural modeling further supported its interaction with viral envelope proteins, while stage-restricted infection assays revealed distinct stages of action: defensin A enhanced adsorption of ZIKV, JEV, and CHIKV, but did not promote DENV-2 adsorption or entry, and instead reduced DENV-2 RNA accumulation at the post-entry replication stage. Our findings highlight a previously unrecognized role of densovirus-derived EVEs in mosquito innate immunity, extending their functional scope from the well-established PIWI-interacting RNA-mediated antiviral defense to the regulation of antimicrobial peptide-associated immune pathways. These findings emphasize the necessity of accounting for EVE activity when analyzing data derived from mosquito cell lines, and suggest that related EVE-mediated immune regulation may contribute to arbovirus dynamics in mosquitoes.IMPORTANCEMosquito-borne viruses such as dengue, Zika, Japanese encephalitis, and chikungunya continue to threaten human health worldwide. Laboratory studies often use Aedes aegypti cell lines to investigate how these viruses interact with their mosquito hosts. Here, we show that the genomes of these cells contain endogenous viral elements derived from mosquito densoviruses. Far from being inert fossils, these sequences are transcriptionally active and regulate mosquito immunity by suppressing the antimicrobial peptide defensin A. This immune modulation influences the replication of different arboviruses in opposite ways, enhancing some while restricting others. Our findings reveal that integrated viral elements can shape the outcome of arbovirus infection, with important implications for interpreting mosquito cell culture experiments and for evaluating endogenous viral element-mediated immune regulation in mosquito-virus interactions.

Aag2 cell↗
PubMed1988

Transposon Tn554 encodes three products required for transposition.

Tn554 is a high-frequency, site-specific, transposable element having integrative properties resembling those of lysogenic bacteriophages. Nucleotide sequence analysis indicates that Tn554 has three transposition genes, designated tnpA, tnpB and tnpC. Mutations in each of these were complemented efficiently in trans by clones containing internal fragments of Tn554; thus the products of these genes function in trans. Elements carrying deletions of the Tn554 termini could not be complemented. The product of tnpC is not absolutely required for transposition, since deletion mutations encompassing 80% of tnpC, as well as frameshift mutations located near the amino terminus of tnpC, transposed at frequencies as high as 2% of that observed with wild-type Tn554. However, such mutations affected the orientation of insertion. With wild-type Tn554 insertion occurs in a single orientation regardless of the orientation of the donor. In tnpC mutants insertion orientation was dictated by the orientation of Tn554 in the donor molecule. A mutant lacking the carboxy-terminal 59 residues of tnpB also exhibited altered insertion orientation. Thus it appears that the tnpC gene product is required for correct orientation of the element upon insertion and that this protein may interact with the carboxy-terminal portion of the tnpB gene product.

Blotting, Southern↗
PubMed1990

Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB.

The mechanism by which the calcium influx signal, triggered by membrane depolarization, is transduced to the nucleus to activate c-fos proto-oncogene transcription has been characterized. A calcium response element (CaRE) that is indistinguishable from a cAMP response element (CRE) mediates transcriptional inducibility by depolarization. Its cognate transcription factor CREB is the target for both calcium and cAMP signals. CREB is rapidly phosphorylated in response to depolarization or cAMP, at a site known to be important for the transcriptional activating function of this protein. The convergent effects of calcium and cAMP on CREB activation are mediated by distinct protein kinase signaling pathways. CREB and its binding site, the Ca/CRE, can thus function as a regulatory element that integrates both calcium and cAMP signals in the control of gene expression.

Adrenal Gland Neoplasms↗
PubMed2005

[Urological attendance in the Integral Health Center "Alto Palancia": model of primary-specialized integration and unique consultation].

INTRODUCTION AND OBJECTIVE: The progressive increase in the demand of urological attendance demands to establish new welfare models that avoid the saturation of consultations and improve the quality in the attention to the user. The objective of this work is to analyze the improvement of the welfare activity developed in Integral the Sanitary Center Alto Palancia, after the restoration of new welfare models based on integration between Attention Primary and Specialized and the putting in practice of the denominated Unique Consultation. MATERIAL AND METHODS: We analyzed the characteristics of the Center, functions and activity of the specialty developed during year 2002. We described to the application of the model of Unique Consultation and the elements of integration with Primary Attention. We analyzed the improvement of the activity through indicators, that we compared with preceding years. In order to determine the degree of satisfaction of the users we made a survey of opinion of patients and/or taken care of relatives in the Center under the new welfare models. Finally we analyzed the advantages that the applied process presents for the patient and the own sanitary institution. RESULTS: 42% of patients have been taken care of by means of the model of Unique Consultation, being the predominant pathology the HBP in the man and the ITUs in the woman. The time of delay for first visit has been reduced from 49 days in single December 2001 to 3 in December 2002. Single 7.7% of patients discharged from the hospital for pursuit by Primary Attention have been sent again to the specialist. The time that the urólogo dedicates to consultations has reduced in a 29%, being this time dedicated to the accomplishment smaller surgery and final reconnaissances (echography, urodynamic studies and others). The opinion survey has shown a satisfaction of the user very elevated. CONCLUSION: The model of Integral Attention Primary-Specialized and the putting in practice of Unique Consultation applicable to the specialty of Urology, increasing the quality perceived by the user and with evident advantages for the Sanitary Organization.

Humans↗
PubMed2005

Assessment of Hansenula polymorpha and Arxula adeninivorans-derived rDNA-targeting elements for the design of Arxula adeninivorans expression vectors.

Different targeting sequences derived from the Arxula adeninivorans and Hansenula polymorpha rDNA clusters were tested in A. adeninivorans integration/expression vectors. For element identification, the rDNA unit of A. adeninivorans (accession number ) was first isolated and characterized in addition to the known H. polymorpha unit. The rDNA is a cluster of some forty 7653-bp units without the 5S rDNA gene. The selected elements were integrated into a set of A. adeninivorans expression/integration vectors harbouring a TEF1 promoter - amyA ORF - PHO5 terminator sequence as reporter gene. No differences in mitotic stability, copy number and transformation frequency were observed. All transformants harboured a single copy integrated into the rDNA by a homologous recombination. In contrast, the choice of the rDNA targeting sequence was found to be of impact on productivity. Use of ETS-18S-5.8S fragments from both organisms resulted in a more than 50% increase in comparison to the use of other elements, independent of the orientation within the vector.

Cloning, Molecular↗
PubMed2005

Genetic structure and chromosomal integration site of the cryptic prophage CP-1639 encoding Shiga toxin 1.

The sequence of 50 625 bp of chromosomal DNA derived from Shiga-toxin (Stx)-producing Escherichia coli (STEC) O111: H- strain 1639/77 was determined. This DNA fragment contains the cryptic Stx1-encoding prophage CP-1639 and its flanking chromosomal regions. The genome of CP-1639 basically resembles that of lambdoid phages in structure, but contains three IS629 elements, one of which disrupts the gene of a tail fibre component. The prophage genome lacks parts of the recombination region including integrase and excisionase genes. Moreover, a capsid protein gene is absent. CP-1639 is closely associated with an integrase gene of an ancient integrative element. This element consists of three ORFs of unknown origin and a truncated integrase gene homologous to intA of CP4-57. By PCR analysis and sequencing, it was shown that this integrative element is present in a number of non-O157 STEC serotypes and in non-STEC strains, where it is located at the 3'-end of the chromosomal ssrA gene. Whereas in most E. coli O111: H- strains, prophages are inserted in this site, E. coli O26 strains contain the integrative element not connected to a prophage. In E. coli O103 strains, the genetic structure of this region is variable. Comparison of DNA sequences of this particular site in E. coli O157: H7 strain EDL933, E. coli O111: H- strain 1639/77 and E. coli K-12 strain MG1655 showed that the ssrA gene is associated in all cases with the presence of foreign DNA. The results of this study have shown that the cryptic prophage CP-1639 is associated with an integrative element at a particular site in the E. coli chromosome that possesses high genetic variability.

Chromosome Mapping↗
PubMed1994

Characterization of transcripts from the Dictyostelium discoideum retrotransposable genetic element DRE.

All of the approximately 150 copies of the Dictyostelium discoideum retrotransposable element DRE are integrated 50 +/- 4 nucleotides upstream from different transfer RNA genes. These genomic regions are generally devoid of informative DNA why integration of the retrotransposon at these position never causes phenotypic mutations. This property makes DRE attractive as a tool in gene therapy where vectors with predictable integration specificities are still not available. Due to the nature as retrotransposon transcription is a prerequisite for the element to integrate at a new genomic position. Here we describe some transcription properties of DRE based on Northern blot analyses and on the characterization of in vitro synthesized cDNAs.

Animals↗