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PubMed1997

Nht1, a transposable element cloned from a dispensable chromosome in Nectria haematococca.

Certain isolates of the plant-pathogenic fungus Nectria haematococca mating population VI (MPVI) contain dispensable chromosomes that are unstable during sexual reproduction. Several of these chromosomes carry genes for phytoalexin detoxification and thus contribute to the pathogenic potential of this organism. A repeated DNA sequence, Nht1, was cloned from one of these dispensable chromosomes in N. haematococca MPVI. One copy of the repeated element (Nht1A) was completely sequenced. It is 2,198 bp long and it possesses incomplete inverted terminal repeats (ITRs) at each end. Nht1B, a partially sequenced copy of Nht1, has complete ITRs. Nht1A appears to contain 2 introns and encodes a protein of 550 amino acids that is highly similar to the protein encoded by the Fusarium oxysporum transposon, Fot1. Due to the presence of ITRs, its repeated nature, and its similarity to Fot1, we conclude that Nht1 is a transposable element. Within North American N. Haematococca MPVI populations, Nht1 is distributed discontinuously. Its copy number in different field isolates varies from zero to approximately 100 copies per genome. The Nht1A source isolate is estimated to contain nine to 11 copies of Nht1; at least six are on the chromosome from which Nht1A was cloned.

Amino Acid Sequence↗
PubMed1986

Viable viruses with deletions in the left inverted terminal repeat define the adenovirus origin of DNA replication.

A series of human adenovirus type 2 genomes with deletions in the left inverted terminal repeat (ITR) have been constructed. Viral genomes that contained a minimum of 45 base pairs (bp) from the terminus of the genome were fully infectious and gave rise to progeny virus which maintained the deletion. In contrast, genomes containing 36 bp or less from the termini of the genome were not infectious. The boundary of a cis-acting element required for viral replication is therefore between 36 and 45 bp from the adenovirus termini and corresponds to the previously identified viral origin of replication, defined using a transfection assay to measure ori activity in vivo. The growth parameters of viruses with deletions in the left ITR were examined. These deletions had no measurable effect on plaque formation or morphology, viral DNA synthesis or early viral mRNA synthesis. Thus, it appears that DNA sequences in the left ITR, outside the replication origin, are completely dispensable for lytic viral growth in tissue culture cells.

Adenoviruses, Human↗
PubMed2002

Homologous recombination between the inverted terminal repeats of defective transposon TCp3.2 causes an inversion in the genome of Cydia pomonella granulovirus.

In this study, a new mutant of the Cydia pomonella granulovirus (CpGV), which shows spontaneous inversion of a transposable element during in vivo replication, is described. CpGV-MCp4 is a natural mutant of CpGV-M, containing the transposable element TCp3.2, which originated from the genome of the host C. pomonella. During in vivo cloning studies of CpGV-MCp4, a mutant called CpGV-MCp4inv was isolated. CpGV-MCp4inv shows heterogeneity in the genome area of transposon insertion. Restriction mapping, PCR analysis and subsequent sequence analysis gave strong evidence that an inversion of TCp3.2 is caused by homologous recombination between the long inverted terminal repeats (ITRs) of the transposon. This finding demonstrated that extensive homologous repeat regions such as the ITRs of transposons cause inversions by homologous recombination during in vivo replication. The observed in vivo inversion between the ITRs can be considered as a model for the contribution of repeated sequences in the genome rearrangement of baculoviruses and a source for genetic heterogeneity among different baculoviruses and baculovirus genotypes.

Animals↗
PubMed2005

Interface thermal resistance between dissimilar anharmonic lattices.

We study interface thermal resistance (ITR) in a system consisting of two dissimilar anharmonic lattices exemplified by the Fermi-Pasta-Ulam and Frenkel-Kontorova models. It is found that the ITR is asymmetric; namely, it depends on how the temperature gradient is applied. The dependence of the ITR on the coupling constant, temperature, temperature difference, and system size is studied. Possible applications in nanoscale heat management and control are discussed.

Journal Article↗
PubMed2005

Susceptibility to antifungal agents of Candida species isolated from paediatric and adult patients with haematological diseases.

Summary The susceptibility to six antifungals: amphotericin B (AMF), 5-fluorocytosine (5-F), miconazole (MIK), ketoconazole (KET), fluconazole (FLU) and itraconazole (ITR) was tested among 206 Candida spp. isolated from paediatric and adult patients with haematological malignancies. To determinate the susceptibility the commercial microdilution method Fungitest (Bio-Rad, France) was used. The strains were classified as susceptible, intermediate susceptible, or resistant on the base of the growth in following breakpoint concentrations of particular drugs: 2 and 8 microg ml(-1) for AMF, 2 and 32 microg ml(-1) for 5-F, 0.5 and 8 microg ml(-1) for MIK, 0.5 and 4 microg ml(-1) for KET and ITR, and 8 and 64 microg ml(-1) for FLU. The highest activity to overall species showed AMF (only one resistant strain) and 5-F (85% susceptible strains). Most of C. albicans isolates were susceptible to tested azoles. The percentages of C. albicans resistant to FLU, ITR, KET and MIK were 4, 11, 8, and 0.8%, respectively. The less susceptible to azoles were C. glabrata and C. krusei (14% and 44% isolates resistant to FLU). A non-albicans Candida isolated from adult patients receiving KET prophylaxis was more frequently resistant to FLU than isolates from patients without previous exposure to azoles (P < 0.05). We did not observe differences in the susceptibility of Candida strains isolated from children compared with those from adults.

Adolescent↗
PubMed1996

Analysis of Hispanic motor vehicle trauma victims in Illinois, 1991-1992.

OBJECTIVE: 1) To develop a profile of Hispanic motor vehicle trauma victims in Illinois, 2) to ascertain whether differences exist between Hispanic and general-Illinois-population motor vehicle trauma victims, and 3) to identify potential target areas for future injury intervention programs. METHODS: A retrospective analysis of Illinois motor vehicle trauma patients admitted from July 1991 to June 1992 was made. Participants were motor vehicle trauma victims (drivers and passengers) who presented to one of 73 level I or level II trauma centers throughout Illinois and were entered into the Illinois Trauma Registry (ITR) from July 1, 1991, through June 30, 1992. RESULTS: Of the 12,299 motor vehicle trauma victims in the ITR, 771 (6.3%) were Hispanic, 8,979 (73.0%) were white, 1,115 (9.1%) were black, and 1,434 (11.6%) were other. When compared with the other racial groups, the group of Hispanic victims were younger (25.2 vs 33.2 years), had higher male predominance (72.8% vs 60.9%), and had the lowest rate of safety equipment/occupant restraint use (21.7% vs 34.7%). A high alcohol use rate (30.7%) and high mean serum ethanol levels (44 mmol/L; 0.2 mg%) were noted. When contrasted with other racial/ethnic groups, the Hispanic victims had lower Injury Severity Scale scores (p < 0.001), but mean hospital charges tended to be higher, with fewer alternative sources of payment (p < 0.001). CONCLUSION: Using age-adjusted data from the ITR, Hispanic motor vehicle trauma victim features differ significantly from those of other racial groups. Effective health maintenance and injury prevention strategies should address the basis for these differences.

Accident Prevention↗
PubMed1998

Disseminated aspergillosis caused by Aspergillus ustus in a patient following allogeneic peripheral stem cell transplantation.

The first case of disseminated aspergillosis caused by Aspergillus ustus in an allogeneic peripheral stem cell transplant patient is described. The patient, a 46-year-old female with a history of myelodysplastic syndrome, underwent high-dose chemotherapy and total body irradiation prior to transplantation. She was released from the hospital 49 days posttransplant (p.t.) in a stable condition with an absolute neutrophil count (ANC) of 2,700 cells per microl. Multiple antimicrobial agents, including itraconazole (ITR), were prescribed during hospitalization and at the time of discharge. Three days after discharge, the patient was readmitted with hemorrhagic cystitis, persistent thrombocytopenia, and bilateral pulmonary consolidation, although no fever was present. The ANC at the time of readmission was 3,500. Upon detection of a pulmonary nodule (day 67 p.t.), a bronchoalveolar lavage was performed; the lavage fluid was positive for both cytomegalovirus and parainfluenza virus and negative for fungus. The patient was placed on ganciclovir. A biopsy specimen from a leg lesion also noted on day 67 p.t. revealed septate hyphae consistent with Aspergillus species, and a culture subsequently yielded Aspergillus ustus. Confirmation detection of A. ustus was made by demonstration of characteristic reproductive structures with the presence of Hülle cells. On day 67 p.t., ITR was discontinued and liposomal amphotericin B (AMB) was initiated. The patient's condition worsened, and she died 79 days p.t. At the time of autopsy, septate hyphae were present in heart, thyroid, and lung tissues, with lung tissue culture positive for A. ustus. In vitro susceptibility testing indicated probable resistance to AMB but not to ITR. This case supports the need for the development of rapid methods to determine antifungal susceptibility.

Aspergillosis↗
PubMed1993

Protein-primed DNA replication: role of inverted terminal repeats in the Escherichia coli bacteriophage PRD1 life cycle.

Escherichia coli bacteriophage PRD1 and its relatives contain linear double-stranded DNA genomes, the replication of which proceeds via a protein-primed mechanism. Characteristically, these molecules contain 5'-covalently bound terminal proteins and inverted terminal nucleotide sequences (inverted terminal repeats [ITRs]). The ITRs of each PRD1 phage species have evolved in parallel, suggesting communication between the molecule ends during the life cycle of these viruses. This process was studied by constructing chimeric PRD1 phage DNA molecules with dissimilar end sequences. These molecules were created by combining two closely related phage genomes (i) in vivo by homologous recombination and (ii) in vitro by ligation of appropriate DNA restriction fragments. The fate of the ITRs after propagation of single genomes was monitored by DNA sequence analysis. Recombinants created in vivo showed that phages with nonidentical genome termini are viable and relatively stable, and hybrid phages made in vitro verified this observation. However, genomes in which the dissimilar DNA termini had regained identical sequences were also detected. These observations are explained by a DNA replication model involving two not mutually exclusive pathways. The generality of this model in protein-primed DNA replication is discussed.

Amino Acid Sequence↗
PubMed1995

Recombinant junctions formed by site-specific integration of adeno-associated virus into an episome.

A model system using an episomal Epstein-Barr virus shuttle vector was recently developed to study the adeno-associated virus (AAV) site-specific integration event in chromosome 19q13.3-qter (C. Giraud, E. Winocour, and K.I. Berns, Proc. Natl. Acad. Sci. USA 91:10039-10043, 1994). In this study, we analyze the recombinant junctions generated after integration of the AAV genome into an Epstein-Barr virus shuttle vector carrying 8.2, 1.6, or 0.51 kb of the chromosome 19 preintegration sequence (AAVS1 locus). In most of the recombinants, one end of the viral genome was joined to a portion of the AAVS1 DNA previously shown to be a minimum target for AAV integration. Within this AAVS1 segment, the AAV insertion points were strikingly clustered around a binding site for the AAV regulatory protein. In all cases, the second junction with AAV occurred with vector DNA outside of the AAVS1 segment. With respect to the viral genome, one junction with the shuttle vector DNA occurred either within the AAV inverted terminal repeat (itr), or near the P5 promoter, approximately 100 nucleotides distal to a modified itr. The modified itr in 5 of 11 recombinants involved a head-to-tail organization. In one such instance, the AAV insert contained slightly more than one genome equivalent arranged in a head-to-tail manner with a junction close to the P5 promoter; the AAV insert in this recombinant episome could be rescued by adenovirus infection and replicated to virus particles. The significance of the head-to-tail organization is discussed in terms of the possible circularization of AAV DNA before or during integration.

Adenoviruses, Human↗
PubMed1997

Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro.

The human parvovirus adeno-associated virus (AAV) is unique in its ability to target viral integration to a specific site on chromosome 19 (ch-19). Recombinant AAV (rAAV) vectors retain the ability to integrate but have apparently lost this ability to target. In this report, we characterize the terminal-repeat-mediated integration for wild-type (wt), rAAV, and in vitro systems to gain a better understanding of these differences. Cell lines latent for either wt or rAAV were characterized by a variety of techniques, including PCR, Southern hybridization, and fluorescence in situ hybridization analysis. More than 40 AAV-rAAV integration junctions were cloned, sequenced, and then subjected to comparison and analysis. In both immortalized and normal diploid human cells, wt AAV targeted integration to ch-19. Integrated provirus structures consisted of head-to-tail tandem arrays with the majority of the junction sequences involving the AAV inverted terminal repeats (ITRs). No complete viral ITRs were directly observed. In some examples, the AAV p5 promoter sequence was found to be fused at the virus-cell junction. Data from dot blot analysis of PCR products were consistent with the occurrence of inversions of genomic and/or viral DNA sequences at the wt integration site. Unlike wt provirus junctions, rAAV provirus junctions mapped to a subset of non-ch-19 sequences. Southern analysis supported the integration of proviruses from two independent cell lines at the same locus on ch-2. In addition, provirus terminal repeat sequences existed in both the flip and flop orientations, with microhomology evident at the junctions. In all cases with the exception of the ITRs, the vector integrated intact. rAAV junction sequence data were consistent with the occurrence of genomic rearrangement by deletion and/or rearrangement-translocation at the integration locus. Finally, junctions formed in an in vitro system between several AAV substrates and the ch-19 target site were isolated and characterized. Linear AAV substrates typically utilized the end of the virus DNA substrate as the point of integration, whereas products derived from AAV terminal repeat hairpin structures in the presence or absence of Rep protein resembled AAV-ch-19 junctions generated in vivo. These results describing wt AAV, rAAV, and in vitro integration junctions suggest that the viral integration event itself is mediated by terminal repeat hairpin structures via nonviral cellular recombination pathways, with specificity for ch-19 in vivo requiring additional viral components. These studies should have an important impact on the use of rAAV vectors in human gene therapy.

Base Sequence↗
PubMed1997

A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle.

Adeno-associated virus (AAV) replication is dependent on two copies of a 145-bp inverted terminal repeat (ITR) that flank the AAV genome. This is the primary cis-acting element required for productive infection and the generation of recombinant AAV (rAAV) vectors. We have engineered a plasmid (pDD-2) containing only 165 bp of AAV sequence: two copies of the D element, a unique sequence adjacent to the AAV nicking site, flanking a single ITR. When assayed in vivo, this modified hairpin was sufficient for the replication of the plasmid vector when Rep and adenovirus (Ad) helper functions were supplied in trans. pDD-2 replication intermediates were characteristic of the AAV replication scheme in which linear monomer, dimer, and other higher-molecular-weight replicative intermediates are generated. Compared to infectious AAV clones for replication, the modified hairpin vector replicated more efficiently independent of size. Further analysis demonstrated conversion of the input circular plasmid to a linear substrate with AAV terminal repeat elements at either end as an initial step for replication. This conversion was independent of both Rep and Ad helper genes, suggesting the role of host factors in the production of these molecules. The generation of these substrates suggested resolution of the modified terminal repeat through a Holliday-like structure rather than replication as a mechanism for rescue. Production of replicative intermediates via this plasmid substrate were competent not only for AAV DNA replication but also for encapsidation, infection, integration, and subsequent rescue from the chromosome when superinfected with Ad and wild-type AAV. These studies demonstrate that this novel 165-bp ITR substrate is sufficient in cis for the AAV life cycle and should provide a valuable reagent for further dissecting the cis sequences involved in AAV replication, packaging, and integration. In addition, this novel plasmid vector can be used as a substrate for both rAAV vector production and synthetic plasmid vector delivery.

DNA, Viral↗
PubMed1999

Factors affecting the terminal resolution site endonuclease, helicase, and ATPase activities of adeno-associated virus type 2 Rep proteins.

The Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) are critical for AAV replication and site-specific integration. They bind specifically to the AAV inverted terminal repeats (ITRs) and possess ATPase, helicase, and strand-specific/site-specific endonuclease activities. In the present study, we further characterized the AAV Rep68/78 helicase, ATPase, and endonuclease activities by using a maltose binding protein-Rep68 fusion (MBP-Rep68Delta) produced in Escherichia coli cells and Rep78 produced in vitro in a rabbit reticulocyte lysate system. We found that the minimal length of single-stranded DNA capable of stimulating the ATPase activity of MBP-Rep68Delta is 100 to 200 bases. The degree of stimulation correlated positively with the length of single-stranded DNA added to the reaction mixture. We then determined the ATP concentration needed for optimal MBP-Rep68Delta helicase activity and showed that the helicase is active over a wide range of ATP concentrations. We determined the directionality of MBP-Rep68Delta helicase activity and found that it appears to move in a 3' to 5' direction, which is consistent with a model in which AAV Rep68/78 participates in AAV DNA replication by unwinding DNA ahead of a cellular DNA polymerase. In this report, we also demonstrate that single-stranded DNA is capable of inhibiting the MBP-Rep68Delta or Rep78 endonuclease activity greater than 10-fold. In addition, we show that removal of the secondary Rep68/78 binding site, which is found only in the hairpin form of the AAV ITR, causes a three- to eightfold reduction in the ability of the ITR to be used as a substrate for the Rep78 or MBP-Rep68Delta endonuclease activity. This suggests that contact between Rep68/78 and this secondary element may play an important role in the Rep-mediated endonuclease activity.

Adenosine Triphosphatases↗
PubMed1999

Isolation of recombinant adeno-associated virus vector-cellular DNA junctions from mouse liver.

Recombinant adeno-associated virus (rAAV) vectors allow for sustained expression of transgene products from mouse liver following a single portal vein administration. Here a rAAV vector expressing human coagulation factor F.IX (hF.IX), AAV-EF1alpha-F.IX (hF.IX expression was controlled by the human elongation factor 1alpha [EF1alpha] enhancer-promoter) was injected into mice via the portal vein or tail vein, or directly into the liver parenchyma, and the forms of rAAV vector DNA extracted from the liver were analyzed. Southern blot analyses suggested that rAAV vector integrated into the host genome, forming mainly head-to-tail concatemers with occasional deletions of the inverted terminal repeats (ITRs) and their flanking sequences. To further confirm vector integration, we developed a shuttle vector system and isolated and sequenced rAAV vector-cellular DNA junctions from transduced mouse livers. Analysis of 18 junctions revealed various rearrangements, including ITR deletions and amplifications of the vector and cellular DNA sequences. The breakpoints of the vector were mostly located within the ITRs, and cellular DNA sequences were recombined with the vector genome in a nonhomologous manner. Two rAAV-targeted DNA sequences were identified as the mouse rRNA gene and the alpha1 collagen gene. These observations serve as direct evidence of rAAV integration into the host genome of mouse liver and allow us to begin to elucidate the mechanisms involved in rAAV integration into tissues in vivo.

Animals↗
PubMed2001

Characterization of adenovirus-induced inverted terminal repeat-independent amplification of integrated adeno-associated virus rep-cap sequences.

Stable packaging cell lines expressing the rep and cap genes for recombinant adeno-associated virus type 2 (rAAV-2) assembly constitute an attractive alternative to transient transfection protocols. We recently characterized a stable HeLa rep-cap cell clone (HeRC32) and demonstrated that upon vector transfection and adenovirus infection, efficient rAAV assembly correlated with a 100-fold amplification of the integrated rep-cap sequence with the inverted terminal repeats (ITRs) deleted. We now report a more detailed analysis of this phenomenon and highlight the key cellular and viral factors involved. Determination of the rep-cap copy number of HeRC32 cells indicated that maximum rep-cap amplification occurred between 24 and 48 h following adenovirus infection. Analysis by pulsed-field gel electrophoresis of adenovirus-infected HeRC32 cells indicated that amplified rep-cap sequences were found in an extrachromosomal form. Amplification of the rep-cap sequence with the ITRs deleted was not dependent on adenovirus replication and still occurred when the highly specific adenovirus polymerase was inactivated. In contrast, amplification was inhibited in the presence of aphidicolin, indicating that cellular polymerases were needed. Our study also documented that among the adenovirus gene products, the DNA-binding protein (DBP) was essential, since rep-cap amplification was severely abrogated when HeRC32 cells were infected at a nonpermissive temperature with an adenovirus mutant encoding a thermosensitive DBP. Furthermore, expression of DBP alone in HeRC32 cells was sufficient to induce a sustained level of rep-cap amplification. Finally, immunofluorescence analysis showed that HeRC32 cells expressing the DBP also simultaneously expressed the Rep proteins, suggesting a possible involvement of the latter in rep-cap amplification. Indeed, the lack of detectable amplification in an adenovirus-infected stable rep-cap HeLa cell clone unable to produce Rep proteins further supported that, among the viral gene products, both the DBP and Rep proteins are necessary to induce the targeted amplification of the integrated rep-cap sequences in the absence of the AAV ITRs.

Adenoviridae↗
PubMed2003

Organization and expression strategy of the ambisense genome of densonucleosis virus of Galleria mellonella.

The expression strategy of parvoviruses of the Densovirus genus has as yet not been reported. Clones were obtained from the densonucleosis virus of Galleria mellonella (GmDNV) that yielded infectious virus upon transfection into LD652 cells. Its genome was found to be the longest (6,039 nucleotides [nt]), with the largest inverted terminal repeats (ITRs) (550 nt) among all parvoviruses. The distal 136 nt could be folded into hairpins with flop or flip sequence orientations. In contrast to vertebrate parvoviruses, the gene cassettes for the nonstructural (NS) and structural (VP) proteins were found on the 5' halves of the opposite strands. The transcripts for both cassettes started 23 nt downstream of the ITRs. The TATA boxes, as well as all upstream promoter elements, were localized in the ITRs and, therefore, identical for the NS and VP transcripts. These transcripts overlapped for 60 nt at the 3' ends (antisense RNAs) at 50 m.u. The NS cassette consisted of three genes of which NS2 was contained completely within NS1 but from a different reading frame. Most of the NS transcripts were spliced to remove the upstream NS3, allowing leaky scanning translation of NS1 and NS2, similar to the genes of RNA-6 of influenza B virus. NS3 could be translated from the unspliced transcript. The VP transcript was not spliced and generated four VPs by a leaky scanning mechanism. The 5'-untranslated region of the VP transcript was only 5 nt long. Despite the transcription and translation strategies being radically different from those of vertebrate parvoviruses, the capsid was found to have phospholipase A(2) activity, a feature thus far unique for parvoviruses.

Amino Acid Sequence↗
PubMed2005

Four-dimensional visualization of the simultaneous activity of alternative adeno-associated virus replication origins.

The adeno-associated virus (AAV) inverted terminal repeats (ITRs) contain the AAV Rep protein-binding site (RBS) and the terminal resolution site (TRS), which together act as a minimal origin of DNA replication. The AAV p5 promoter also contains an RBS, which is involved in Rep-mediated regulation of promoter activity, as well as a functional TRS, and origin activity of these signals has in fact been demonstrated previously in the presence of adenovirus helper functions. Here, we show that in the presence of herpes simplex virus type 1 (HSV-1) and AAV Rep protein, p5 promoter-bearing plasmids are efficiently amplified to form large head-to-tail concatemers, which are readily packaged in HSV-1 virions if an HSV-1 DNA-packaging/cleavage signal is provided in cis. We also demonstrate simultaneous and independent replication from the two alternative AAV replication origins, p5 and ITR, on the single-cell level using multicolor-fluorescence live imaging, a finding which raises the possibility that both origins may contribute to the AAV life cycle. Furthermore, we assess the differential affinities of Rep for the two different replication origins, p5 and ITR, both in vitro and in live cells and identify this as a potential mechanism to control the replicative and promoter activities of p5.

Animals↗
PubMed1986

Template requirements for in vivo replication of adenovirus DNA.

The adenovirus (Ad) DNA origin of replication was defined through an analysis of the DNA sequences necessary for the replication of plasmid DNAs with purified viral and cellular proteins. Results from several laboratories have shown that the origin consists of two functionally distinct domains: a 10-base-pair sequence present in the inverted terminal repetition (ITR) of all human serotypes and an adjacent sequence constituting the binding site for a cellular protein, nuclear factor I. To determine whether the same nucleotide sequences are necessary for origin function in vivo, we developed an assay for the replication of plasmid DNAs transfected into Ad5-infected cells. The assay is similar to that described by Hay et al. (J. Mol. Biol. 175:493-510, 1984). With this assay, plasmid DNA replication is dependent upon prior infection of cells with virus and only occurs with linear DNA molecules containing viral terminal sequences at each end. Replicated DNA is resistant to digestion with lambda-exonuclease, suggesting that a protein is covalently bound at both termini. A plasmid containing only the first 67 base pairs of the Ad2 ITR replicates as well as plasmids containing the entire ITR. Deletions or point mutations which reduce the binding of nuclear factor I to DNA in vitro reduce the efficiency of plasmid replication in vivo. A point mutation within the 10-base-pair conserved sequence has a similar effect upon replication. These results suggest that the two sequence domains of the Ad origin identified by in vitro studies are in fact important for viral DNA replication in infected cells. In addition, we found that two separate point mutations which lie outside these two sequence domains, and which have little or no effect upon DNA replication in vitro, also reduce the apparent efficiency of plasmid replication in vivo. Thus, there may be elements of the Ad DNA origin of replication which have not yet been identified by in vitro studies.

Adenoviruses, Human↗
PubMed2005

Relationships between physiological variables and race performance in French standardbred trotters.

The relationships between V4 (the velocity for a blood lactate concentration of 4 mmol/litre) and V200 (the velocity for a heart rate of 200 bpm) and the performance indices of 223 healthy French trotters were analysed. They were divided into four age groups of three, four, five and six years and over, and into three performance groups (good, intermediate and poor performers) defined by the index of trot (ITR), an annual index of performance calculated by the national stud. V4 and V200 were assessed during the performance of a three-step standardised field exercise test, and four performance indexes (ITR, earnings, best time and the number of starts) were calculated for each group. An analysis of variance showed that V4 and V200 were highly correlated with the horses' age and level of performance (P<0.05). The correlation between the physiological variables and ITR was better for the three- and four-year-old horses than for the older horses.

Age Factors↗