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Identification of a novel family of human endogenous retroviruses and characterization of one family member, HERV-K(C4), located in the complement C4 gene cluster.

We have identified a novel family of about 10-50 human endogenous retrovirus elements (HERVs) and have characterized one family member (HERV-KC4). This retrovirus element is integrated within intron 9 of and complement C4A genes and also in some C4B genes, and is a principal contribution to interlocus and interallelic length heterogeneity of C4 genes. The HERV-K(C4) sequence has a typical retrovirus structure with elements of gag, pol and env domains, flanked by two long terminal repeats (LTRs) and is similar to type A, B and D retroviruses. Multiple termination codons preclude the existence of long open reading frames, suggesting that the HERV-K(C4) sequence is no longer functional. Zoo blot hybridization reveals that New World monkeys appear to lack sequences similar to HERV-K(C4), suggesting that integration has occurred after the divergence of Old and New World monkeys. Retrotransposition of prototype viruses is presumed to have led to the amplification and integration of the members of the family in different loci, which in humans, appear to be dispersed over several chromosomes. The absence of the HERV-K(C4) element in some C4B genes in both humans and orangutangs indicate that the retrovirus inserted into the C4A gene after the duplication of the cluster. Subsequent spread of the HERV-K(C4) sequence to C4B genes presumably occurred by interlocus sequence exchange mechanisms, such as unequal crossover and gene conversion-like mechanisms.

Animals↗

A nuclear protein factor binds specifically to the 3'-regulatory module of the long-interspersed-nuclear-element-like Dictyostelium repetitive element.

The Dictyostelium repetitive element DRE integrates in a position-specific manner upstream of tRNA genes in the Dictyostelium discoideum genome. DRE has structural similarities to the group of long interspersed nuclear elements, whose replication mechanism is poorly understood. The C-module at the 3' end of DRE encodes a regulatory cis-acting sequence that contains an RNA polymerase II promoter. This promoter directs the synthesis of RNAs that are thought to play a critical role in DRE transposition. In this study, we describe the identification of a nuclear protein factor that binds to the C-module in a sequence-specific manner. The C-module-binding factor (CMBF) recognizes three DNA sequence motifs that contain homopolymeric (dT) stretches of variable lengths, but does not bind to a standard RNA polymerase II promoter from D, discoideum. Analysis of highly CMBF-enriched fractions and glycerol gradient sedimentation of CMBF suggest that the factor exists as a monomeric 115-kDa protein. Possible roles of CMBF in DRE transposition are discussed.

Animals↗

[Response of pituitary and adrenal elements in a state of cardiorespiratory system integration].

Morpho-functional equivalents of reaction of the hypophyseal and adrenal elements have been studied in 7 test and 10 control dogs biometrically, histologically, histoenzymatically and electron microscopically under the condition of integration in the cardio-respiratory systems. The condition has been modelled by individually chosen physical loading (running in the treadmill) up to the IV stage of the organism's adaptation according to the author's technique. The integration state is developing instead of the starting desintegration state (the beginning of the work) and is characterized by an economical and effective functioning of the cardio-respiratory system elements. At the integration of the cardio-respiratory apparatus, the hypophysial-adrenal system is activating. The principle of the zonal reaction in the adrenals and that of the selective activity of the adenohypophysis is clearly followed, that is demonstrated in the predominant activation of the bundle zone elements, chromophobic and basophilic adenocytes where certain signs of the secretory excretion predominate. In cells of the glomerular zone the signs of synthetic processes prevail over those of the secretory excretion. In cells of the medullary substance the signs of the preceding forced secretory excretion are clearly seen. A suggestion is made that it is expedient to elaborate training regimes according to the loading determining integration of the organism's cardio-respiratory systems.

11-Hydroxycorticosteroids↗

Integration of a vector containing rodent repetitive elements in the rat genome.

We have previously shown that integration of a polyoma vector containing rodent repetitive elements into rat cellular DNA is non-random (Wallenburg et al. J. Virol. 50: 678-683). Junctions between the polyoma vector and the host DNA occur in the repetitive sequences of the vector about ten times more frequently than would be expected if sequences from the vector were used randomly for integration. In this paper we looked at the host sequences involved in these junctions. Our analysis did not reveal any repetitive or specific sequences and we presume therefore that the repetitive sequences of the vector acted as hot spots for illegitimate recombination. We also analysed the integration mechanism and found that: First, even though the polyoma vector was transfected in the presence of carrier DNA, integration did not involve the formation of a transgenome. Second, in at least one of the clones analysed, integration resulted in deletion of host DNA sequences. Third, the host DNA displaced at the integration site was considerably longer than the integrated segment.

Animals↗

Analytic environment-dependent tight-binding bond integrals: application to MoSi2.

We present the first derivation of explicit analytic expressions for the environmental dependence of the sigma, pi, and delta bond integrals within the orthogonal two-center tight-binding approximation by using the recently developed bond-order potential theory to invert the nonorthogonality matrix. We illustrate the power of this new formalism by showing that it not only captures the transferability of the bond integrals between elemental bcc Mo and Si and binary C11(b) MoSi2 but also predicts the absence of any discontinuity between first and second nearest neighbors for the ddsigma bond integral even though large discontinuities exist for ppsigma, pppi, and ddpi.

Journal Article↗

Institutional integrity in Roman Catholic health care institutions.

Issues of institutional identity and integrity in Roman Catholic health care institutions have been addressed at the level of individual institutions as well as by organizations of Catholic health care providers and at various levels in the Church hierarchy. The papers by Carol Taylor, C.S.F.N., Thomas Shannon, Kevin O'Rourke, O.P., Gerard Magill in this volume provide a significant contribution to concerns of Roman Catholic health care institutions as they face the challenges of providing health care in a secular, pluralistic, market-driven economy. One way to understand institutional integrity is as a measure of the coherence between what an institution identifies as its commitments (its stated moral character), what an institution does (its manifest moral character) and an institution's fundamental moral commitments (its deep moral character). The essays in this volume support this model of integrity. Although it is not their explicit focus, the four essays together provide a vision of institutional integrity for Catholic health care institutions. Each author focuses on one of the three central aspects of integrity: what one identifies as one's commitments (Taylor), how one's actions reflect one's values (Shannon and Magill), and what one is or what one values at a deep level (O'Rourke). I will offer a brief overview of the ways in which the integrity of Catholic health care institutions has been addressed. Then I will consider the four essays and show how each offers an analysis of one of the three critical elements of integrity.

Catholicism↗

Template jumping by a LINE reverse transcriptase has created a SINE-like 5S rRNA retropseudogene in Dictyostelium.

Short interspersed nuclear elements (SINEs) are non-autonomous retroelements that mimic the 3' ends of so-called long interspersed nuclear elements (LINEs) to ensure their propagation by proteins encoded by autonomous LINEs. The Dictyostelium discoideum genome contains a family of LINE-like retrotransposons that specifically target tRNA genes for integration (TRE elements). We describe here a retrotransposed ribosomal 5S RNA pseudogene in the D. discoideum genome that contains at its 3' end an 8-bp sequence derived from the 3' end of a TRE and a polyadenine tail. The r5S "retropseudogene" is flanked by target-site duplications that are characteristic for TREs, and is inserted upstream of a tRNA gene, just like a typical TRE. The D. discoideum r5S retropseudogene has structural features of a SINE, but has not been amplified, probably due to the 5'-truncation that occurred upon its initial retrotransposition. The discovery of this D. discoideum r5S retropseudogene reveals that SINEs can be created de novo during reverse transcription of LINE transcripts, if the LINE-encoded reverse transcriptase dissociates from the LINE RNA and jumps to other cellular RNAs-particularly genes transcribed by RNA polymerase III-to create continuous mixed cDNAs.

Animals↗

Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC.

Staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element composed of the mec gene complex, which encodes methicillin resistance, and the ccr gene complex, which encodes the recombinases responsible for its mobility. The mec gene complex has been classified into four classes, and the ccr gene complex has been classified into three allotypes. Different combinations of mec gene complex classes and ccr gene complex types have so far defined four types of SCCmec elements. Now we introduce the fifth allotype of SCCmec, which was found on the chromosome of a community-acquired methicillin-resistant Staphylococcus aureus strain (strain WIS [WBG8318]) isolated in Australia. The element shared the same chromosomal integration site with the four extant types of SCCmec and the characteristic nucleotide sequences at the chromosome-SCCmec junction regions. The novel SCCmec carried mecA bracketed by IS431 (IS431-mecA-DeltamecR1-IS431), which is designated the class C2 mec gene complex; and instead of ccrA and ccrB genes, it carried a single copy of a gene homologue that encoded cassette chromosome recombinase. Since the open reading frame (ORF) was found to encode an enzyme which catalyzes the precise excision as well as site- and orientation-specific integration of the element, we designated the ORF cassette chromosome recombinase C (ccrC), and we designated the element type V SCCmec. Type V SCCmec is a small SCCmec element (28 kb) and does not carry any antibiotic resistance genes besides mecA. Unlike the extant SCCmec types, it carries a set of foreign genes encoding a restriction-modification system that might play a role in the stabilization of the element on the chromosome.

Amino Acid Sequence↗

Efficient integration of recombinant adeno-associated virus DNA vectors requires a p5-rep sequence in cis.

The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10- to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5-rep promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.

Adenoviruses, Human↗

Sequence-specific DNA binding by p53: identification of target sites and lack of binding to p53 - MDM2 complexes.

An immune selection procedure was employed in order to isolate p53 binding sites from mouse genomic DNA. Two DNA clones capable of tight specific interaction with wild type p53 were subjected to further characterization. In both cases, the p53 binding regions displayed a high degree of sequence homology with the consensus binding site defined for human genomic DNA. One of the clones was found to be derived from the LTR of a retrovirus-like element (a member of the GLN family). The region encompassing the GLN LTR p53 binding site could confer p53 responsiveness upon a heterologous promoter. Furthermore, the expression of the endogenous, chromosomally integrated GLN elements was significantly induced upon activation of wild type p53 in cells harboring a temperature sensitive p53 mutant. Finally, it was demonstrated that p53 - MDM2 complexes fail to bind tightly to such a p53 binding site. This may contribute to the inhibition by MDM2 of p53-mediated transcriptional activation.

Animals↗

Pre-exposure to UV irradiation increases the transfer frequency of the IncJ conjugative transposon-like elements R391, R392, R705, R706, R997 and pMERPH and is recA+ dependent.

The enteric conjugative transposon-like IncJ elements R391, R392, R705, R706 and pMERPH, all demonstrated increased conjugative transfer upon UV irradiation. The transfer frequency increased on average from its basal rate of 10(-5) to 10(-3) per recipient, upon pre-exposure to UV irradiation. However, the transfer frequency of R997, which was higher than the other IncJ elements at 10(-3) per donor, showed a smaller increase. This effect was shown to be recA+ dependent in all cases. Using PCR primers directed outwards from the ends of the integrated R391 element it was observed that a circular intermediate of the element forms within the host, which has been proposed to be a transfer intermediate. Using real-time PCR, it was determined that the amount of the circular intermediate produced increased substantially upon UV irradiation.

Conjugation, Genetic↗

Extrachromosomal copies of transposon Tc1 in the nematode Caenorhabditis elegans.

Extrachromosomal copies of the 1.6-kilobase transposable element Tc1 are present at the level of between 0.1 and 1.0 copy per cell in Caenorhabditis elegans strain Bergerac. Extrachromosomal elements were detected and studied using Southern hybridizations employing a Tc1-specific probe. The amount of extrachromosomal Tc1 DNA was roughly constant during development in Bergerac, which has approximately 300 integrated chromosomal copies of Tc1 in its haploid genome. Extrachromosomal Tc1 DNA was not detected in strain Bristol, which has 30 chromosomal copies of Tc1. Three forms of extrachromosomal DNA were detected. The predominant form was a 1.6-kilobase linear molecule with ends corresponding to the ends of an integrated Tc1 element. The other two forms were, respectively, relaxed and supercoiled circular copies of the element. Structural assignments were based on electrophoretic mobility, the results of sedimentation velocity and equilibrium density gradient experiments, and on the sizes of the products produced by treatment of purified extrachromosomal DNA with restriction endonucleases. The suggestion is made that these extrachromosomal transposable elements are the products of excision events known to be occurring at high frequency in somatic cells in Bergerac.

Animals↗

Unusual structure of the attB site of the site-specific recombination system of Lactobacillus delbrueckii bacteriophage mv4.

The temperate phage mv4 integrates its genome into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus by site-specific recombination within the 3' end of a tRNA(Ser) gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage attP site and the bacterial attB site. In this study, we show that the mv4 integrase functions in vivo in Escherichia coli and we characterize the bacterial attB site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the attB sequence was determined by site-directed mutagenesis. The structure of the attB site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNA(Ser) gene were present within the minimal attB site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model.

Attachment Sites, Microbiological↗

Genome analysis of an inducible prophage and prophage remnants integrated in the Streptococcus pyogenes strain SF370.

The mitomycin C inducible prophage SF370.1 from the highly pathogenic M1 serotype Streptococcus pyogenes isolate SF370 showed a 41-kb-long genome whose genetic organization resembled that of SF11-like pac-site Siphoviridae. Its closest relative was prophage NIH1.1 from an M3 serotype S. pyogenes strain, followed by S. pneumoniae phage MM1 and Lactobacillus phage phig1e, Listeria phage A118, and Bacillus phage SPP1 in a gradient of relatedness. Sequence similarity with the previously described prophages SF370.2 and SF370.3 from the same polylysogenic SF370 strain were mainly limited to the tail fiber genes. As in these two other prophages, SF370.1 encoded likely lysogenic conversion genes between the phage lysin and the right attachment site. The genes encoded the pyrogenic exotoxin C of S. pyogenes and a protein sharing sequence similarity with both DNases and mitogenic factors. The screening of the SF370 genome revealed further prophage-like elements. A 13-kb-long phage remnant SF370.4 encoded lysogeny and DNA replication genes. A closely related prophage remnant was identified in S. pyogenes strain Manfredo at a corresponding genome position. The two prophages differed by internal indels and gene replacements. Four phage-like integrases were detected; three were still accompanied by likely repressor genes. All prophage elements were integrated into coding sequences. The phage sequences complemented the coding sequences in all cases. The DNA repair genes mutL and mutS were separated by the prophage remnant SF370.4; prophage SF370.1 and S. pneumoniae phage MM1 integrated into homologous chromosomal locations. The prophage sequences were interpreted with a hypothesis that predicts elements of cooperation and an arms race between phage and host genomes.

Attachment Sites, Microbiological↗

Zebrafish genetics: harnessing horizontal gene transfer.

The promiscuous spread of Tc1/mariner transposons across species implies that host factors are relatively unimportant for their transposition. Heterologous elements can integrate on expression of the corresponding transposases, an approach that should greatly facilitate genetic analysis in the zebrafish.

Animals↗

The structure of hobo transposable elements and their insertion sites.

The hobo transposable elements of Drosophila form a family of 3.0-kb elements and their deletion derivatives. Their distribution is consistent with the model that 3.0-kb elements are functionally complete but that smaller hobos are defective and require complete elements in trans for transposition. The sequence of one 3.0-kb element is presented; it has several interesting features, including a 1.9-kb open reading frame downstream from potential TATA and CAT sequences. Comparison of 11 independent insertion sites shows that in every case the hobo element has integrated at and duplicated either the sequence NNNNNNAC or CTTTNNNN. There is evidence that an eight nucleotide sequence internal to hobo that matches both of these sequences has been used as an insertion site for a second hobo element, as the first step in the creation of an internal deletion derivative. Structural similarities between hobo and the eukaryotic transposable elements P, Ac, 1723, and Tam3, found in widely divergent host organisms, suggest that they all transpose by a common mechanism.

Journal Article↗

Mechanisms of noise-resistance in genetic oscillators.

A wide range of organisms use circadian clocks to keep internal sense of daily time and regulate their behavior accordingly. Most of these clocks use intracellular genetic networks based on positive and negative regulatory elements. The integration of these "circuits" at the cellular level imposes strong constraints on their functioning and design. Here, we study a recently proposed model [Barkai, N. & Leibler, S. (2000) Nature (London), 403, 267-268] that incorporates just the essential elements found experimentally. We show that this type of oscillator is driven mainly by two elements: the concentration of a repressor protein and the dynamics of an activator protein forming an inactive complex with the repressor. Thus, the clock does not need to rely on mRNA dynamics to oscillate, which makes it especially resistant to fluctuations. Oscillations can be present even when the time average of the number of mRNA molecules goes below one. Under some conditions, this oscillator is not only resistant to but, paradoxically, also enhanced by the intrinsic biochemical noise.

Algorithms↗

Cytoskeletal and muscle-like elements in cochlear hair cells.

Monospecific antibodies to actin and to tubulin were used as immunofluorescent probes to evaluate the distribution of microtubules and actin filaments in the organ of Corti in mouse and guinea pig. The results indicate that in cochlear receptor cells actin and actin filaments as well as tubulin and microtubules are integral cytoskeletal elements. The presence of actin suggests a possible contractile mechanism within the sensory cilia whereas tubulin is thought to play an important role in the stability of sensory cells. Both proteins are discussed to form structural elements required for the mechano-chemical coupling in hearing.

Actins↗