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At least 667 records · Page 37Linked to original sources
PubMed1986

Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K.

The three replication origins of the antibiotic resistance plasmid R6K require for their activity in Escherichia coli a DNA segment containing seven 22 base-pair direct repeats and a plasmid-encoded initiation protein (pi). The pi protein functions in the negative control of R6K replication, in addition to its requirement for the initiation of replication. Construction of a plasmid containing the pi structural gene (pir) downstream from the inducible pR promoter of bacteriophage lambda provided high levels of production of pi protein in E. coli. The pi protein was purified and shown to possess general DNA binding properties with a preference for DNA fragments containing the gamma origin of replication, the operator region of the pir gene and the R6K beta-origin region. Velocity sedimentation analysis indicates that the pi protein exists as a dimer in its native form. Agarose gel electrophoresis analysis of pi-gamma-origin complexes suggests that one pi dimer binds to each copy of the 22 base-pair direct repeats in the gamma origin region. Purified mutant pi protein obtained from a temperature-sensitive initiation mutant (pir 105-ts) exhibited temperature-sensitive binding activity to the gamma-origin region, whereas two mutant proteins exhibiting a high copy number phenotype were unaltered (pir104-cop) or slightly reduced (pir1-cop) in binding activity. The patterns of DNase I protection and enhancement were similar for the wild-type and mutant proteins examined.

Binding Sites↗
PubMed1988

Methylation is co-ordinated on the putative replication origins of Physarum ribosomal DNA.

In Physarum polycephalum, the ribosomal DNA is found as 60,000 base-pair palindromes. Each rDNA has four symmetrically arranged replication origins flanked by ribosomal RNA genes. A particular sequence, the putative replication origin, is repeated at the approximate position of each origin and nowhere else in the molecule. On a typical rDNA molecule, only one origin is active per replication cycle. We show that both the level and co-ordination of methylation result in asymmetrically methylated rDNA molecules that are particularly hypomethylated at one of their four putative replication origins. This pattern of methylation on a typical rDNA molecule is consistent with a model where hypomethylation is a determinant of origin activity.

Cell Cycle↗
PubMed1988

Initiation of DNA replication at cloned origins of bacteriophage T7.

Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by an A + T-rich region. When the primary origin region is deleted replication initiates at secondary origins. We have analyzed the ability of plasmids containing cloned fragments of T7 to replicate after infection of Escherichia coli with bacteriophage T7. All cloned T7 fragments that support plasmid replication contain a T7 promoter but a T7 promoter alone is not sufficient for replication. Replication of plasmids containing the primary origin is dependent on T7 DNA polymerase and gene 4 protein (helicase/primase) and a portion of the A + T-rich region. The other T7 fragments that support plasmid replication after T7 infection are promoter regions phi OR, phi 13 and phi 6.5 (secondary origins). When both the primary and secondary origins are present simultaneously on compatible plasmids, replication of each is temporally regulated. Such regulation may play a role during T7 DNA replication.

Base Sequence↗
PubMed1988

Protein-induced bending of the simian virus 40 origin of replication.

A 3.5 S protein, isolated from mammalian nuclei, specifically binds to DNA fragments containing the simian virus 40 (SV40) origin of replication. Two distinct nucleoprotein complexes are formed, a complex with high electrophoretic mobility carrying probably only one protein molecule, and a complex with reduced electrophoretic mobility carrying probably two protein molecules per DNA fragment. Band shift competition as well as methylation interference assays locate the binding site of the protein in the A + T-rich "late" region of the origin between SV40 nucleotides 13 and 35. The late origin binding (LOB) protein and T antigen bind simultaneously to adjacent sites in the origin. Using circularly permuted DNA fragments of identical lengths we show that the LOB protein induces pronounced bending of the origin fragment. The bending center maps at the 5' end of the adenine tract with one bound protein molecule and at the 3' end when two LOB proteins are bound to one origin fragment.

Antigens, Viral, Tumor↗
PubMed1985

Comparison of imipramine-imipraminium in mice. To elucidate central or peripheral origin of effects of imipramine.

Imipramine hydrochloride shows effects in a battery of tests used for the screening of antidepressant drugs. The central origin of these pharmacological effects of imipramine has not been clearly established. Imipramine methiodide is a quaternary derivative of imipramine which does not cross the blood-brain barrier easily. The effects of the two forms of imipramine have been compared: on an effect known to have a central origin; on two effects known to have a peripheral origin; on a battery of tests used for the screening of antidepressant drugs. It has been demonstrated that imipramine methiodide is as active as imipramine hydrochloride on two effects of peripheral origin, less active than imipramine hydrochloride on an effect considered to have a central origin and less active than imipramine hydrochloride or inactive on the tests which are used for the screening of antidepressant drugs. Consequently, the tests used for the screening of antidepressant drugs represent, primarily or exclusively, effects of central origin.

Animals↗
PubMed1985

Expression of an immediate early polypeptide and activation of a viral origin of DNA replication in cells containing a fragment of herpes simplex virus DNA.

A thymidine kinase cotransformation procedure has been used to introduce the sequences encoding the herpes simplex virus type 1 (HSV-1) immediate early protein, Vmw175, into permissive cells either in the presence or the absence of the adjacent origin of viral DNA replication. Cells transformed by either origin-plus or origin-minus DNA were capable of expressing functional Vmw175 as indicated by their ability to complement the growth at the nonpermissive temperature of an HSV-1 mutant, ts K, containing a temperature-sensitive lesion in the Vmw175 gene. A proportion of the virus yield from cells transformed with the origin-plus, but not the origin-minus, plasmid exhibited a ts+ phenotype. The generation of ts+ virus correlated with an amplification of input plasmid DNA sequences which occurred following superinfection, suggesting that recombination between the ts mutant and the amplified viral DNA sequences had taken place. Encapsidation of the amplified DNA sequences was also detected, suggesting that in addition to a functional origin of replication and Vmw175 gene the transformed cells also retain the viral DNA packaging signals.

Capsid↗
PubMed1990

Effect of human cytomegalovirus on replication of SV40 origin and the expression of T antigen.

Human cytomegalovirus (HCMV) induces replication of the cloned SV40 origin of DNA replication in both productive and nonproductive infections. HCMV-induced replication of the SV40 DNA origin required the presence of T antigen. Human embryonic lung (HEL) cells were found to be fully permissive for SV40 origin replication only in the presence of HCMV gene expression. In addition, expression of plasmid encoded SV40 T antigen in HEL cells was only induced in the presence of HCMV. Cotransfection of the SV40-cloned origin of replication and seven overlapping cosmids containing the entire HCMV genome is capable of stimulating replication of the SV40 origin. We conclude that induction of the SV40 origin of replication by HCMV is (i) not due to any component of the HCMV virion, (ii) has a requirement for T antigen expression, (iii) occurs in an SV40 nonproductive cell, when T antigen expression is induced by HCMV gene expression.

Animals↗
PubMed1985

Conformational changes in a replication origin induced by an initiator protein.

The replication initiator protein of the plasmid R6K binds to seven contiguous 22 bp direct repeats that form an indispensable part of the three replication origins alpha, beta, and gamma. Binding of the initiator to the direct repeats induced a marked bending of the region of gamma replication origin. Binding of the initiator also promoted unwinding of the origin DNA by at least two turns. Distamycin appeared to antagonize the binding of the initiator to the seven 22 bp direct repeats. At the appropriate DNA and protein concentrations the initiator enhanced topoisomerase-induced catenation of the origin containing supercoiled DNA but not of DNA lacking the origin sequence. Thus, the initiator protein caused significant changes in the secondary and tertiary structures of the replication origin.

Bacterial Proteins↗
PubMed1992

Gene amplification in a human osteosarcoma cell line results in the persistence of the original chromosome and the formation of translocation chromosomes.

Although gene amplification, a process that is markedly enhanced in tumor cells, has been studied in many different cell systems, there is still controversy about the mechanism(s) involved in this process. It is still unclear what happens to the DNA sequences that become amplified, whether they remain present at their original location (conservative gene amplification) or whether gene amplification necessarily results in a deletion at the original location (non-conservative gene amplification). We have studied gene amplification in a human osteosarcoma cell line, starting from a cell clone which contains only one copy of a plasmid integrate. Independent amplificants, originating from this clone and containing elevated plasmid copy numbers, were isolated and analyzed. Based on previous observations, encompassing the persistence of single-copy DNA sequences besides amplified DNA sequences clustered at a different location in the independent amplificants, we proposed an amplification pathway including a local duplication step and transposition of the duplicated DNA to other chromosomal positions. Now we have extended our study to more independent amplificants. We prove that the single-copy plasmid-containing chromosomes in the different amplificants and the single-copy plasmid-containing chromosome in the original parental cell clone are indeed identical, namely a translocation chromosome composed of at least three parts of which two originate from chromosomes 14 and 17. We show that the unit of amplification and the unit of the proposed transposition event are at least 1.5 Mb. We also demonstrate that the amplified DNA sequences, present at genomic locations other than the original single-copy DNA sequences, are preferentially associated with chromosome 16. We find that the amplified DNA sequences are often located at or near a site of chromosome translocation involving chromosome 16. In one cell clone we detect the amplified DNA sequences in most of the cells to be located within a complete chromosome 16 while in a minority of cells the amplified sequences are located at or near a breakpoint on a translocation chromosome 16. This indicates that this amplification region is highly unstable and frequently gives rise to translocation events.

Blotting, Southern↗
PubMed1988

Protein-DNA interactions at the simian virus 40 origin of replication.

Simian Virus 40 (SV40)-encoded large T antigen has an intrinsic ATP-dependent DNA-unwinding activity which is necessary for an early step in the activation of the viral origin of replication. Isolated T antigen unwinds any double-stranded DNA, regardless of whether it is linear or circularly closed. However, initiation of DNA replication depends on an intact origin of replication, and even minor deviations from the wild-type origin sequence abolish the template activity of an origin-bearing plasmid. This discrepancy suggests that T antigen may not be sufficient for origin activation and that other, probably cellular, functions are involved. We have isolated a cellular protein, the LOB protein, which specifically interacts with the AT-rich region of the SV40 origin and which induces a pronounced bending of the bound DNA.

Antigens, Polyomavirus Transforming↗
PubMed1991

Enhancement of DNA replication by transcription factors NFI and NFIII/Oct-1 depends critically on the positions of their binding sites in the adenovirus origin of replication.

The origin of DNA replication of many human adenoviruses is composed of a highly conserved core origin and an auxiliary region, containing the binding sites for NFI and NFIII/Oct-1. We examined enhancement of DNA replication in vitro by the purified functional DNA-binding domains of NFI (NFI-BD) and NFIII/Oct-1 (the POU domain), using origins in which the positions of the binding sites for these proteins were transposed. Insertion or deletion of two or three base pairs between the core origin and the NFI binding site resulted in a 3-5-fold decrease of stimulation, whereas larger insertions gradually reduced the stimulation further. Mutants in which the NFI binding site was separated approximately one or two helical turns from the core origin by AT-rich sequences could still be stimulated by NFI. In contrast, insertion of two or more base pairs between the NFI and NFIII/Oct-1 binding sites abolished stimulation by NFIII/Oct-1 almost completely. Furthermore, stimulation by this protein was lost when the Ad2 NFIII/Oct-1 binding site was transposed to a position closer to the core origin, destroying the NFI binding site. This shows that the position of the NFIII/Oct-1 binding site is essential for stimulation. Models to explain these position-dependent effects on stimulation are discussed.

Adenoviridae↗
PubMed1992

The number of symbiotic origins of organelles.

Mitochondria and chloroplasts both originated from bacterial endosymbionts. The available evidence strongly supports a single origin for mitochondria and only somewhat less strongly a single, slightly later, origin for chloroplasts. The arguments and evidence that have sometimes been presented in favor of the alternative theories of the multiple or polyphyletic origins of these two organelles are evaluated and the kinds of data that are needed to test more rigorously the monophyletic theory are discussed. Although chloroplasts probably originated only once, eukaryotic algae are polyphyletic because chloroplasts have been secondarily transferred to new lineages by the permanent incorporation of a photosynthetic eukaryotic algal cell into a phagotrophic protozoan host. How often this has happened is much less clear. It is particularly unclear whether or not the chloroplasts of typical dinoflagellates and euglenoids originated in this way from a eukaryotic symbiont: their direct divergence from the ancestral chloroplast cannot be ruled out and indeed has several arguments in its favor. The evidence for and against the view that the chloroplast of the kingdom Chromista was acquired in a single endosymbiotic event is discussed. The possibility that even the chloroplast of Chlorarachnion might have been acquired during the same symbiosis that created the cryptomonad cell, if the symbiont was a primitive alga that had chlorophyll a, b and c as well as phycobilins, is also considered. An alga with such a combination of pigments might have been ancestral to all eukaryote algae.

Biological Evolution↗
PubMed1986

Efferent projections of nucleus locus coeruleus: topographic organization of cells of origin demonstrated by three-dimensional reconstruction.

The present study examines the spatial distribution within rat locus coeruleus of neurons projecting to particular brain regions. In order to accurately recreate, in digital and pictorial formats, the spatial distribution of neurons for the entire nucleus locus coeruleus, three-dimensional reconstructions were created which specified the location of each individual Nissl-stained locus coeruleus cell in each of five nuclei. Dynamic computerized displays were visually analyzed and statistically compared. The nuclei from different brains were found to be strikingly similar in density and distribution of cells. In order to determine whether the cells of origin for particular locus coeruleus projections were clustered within the nucleus, reconstructions were created of the distributions of cells labeled by injections of a retrograde tracer, horseradish peroxidase, into particular terminal regions. Groups consisting of animals with injections into the same target areas were visually and statistically compared. The cells of origin of most efferent projections were found to be spatially organized within locus coeruleus. Specifically, projections to both the dorsal and ventral hippocampus originated solely from the dorsal segment of the nucleus, while spinal cord projections originated from ventral-posterior locus coeruleus. Cells of origin of cerebral and cerebellar cortical efferents, as well as hypothalamic efferents, exhibited less clustering, although reliable differences in distribution were observed. The most striking example of clustered cells of origin was exhibited by the large norepinephrine-containing cells constituting the anterior pole of locus coeruleus which were labeled only by hypothalamic injections. This analysis of spatial organization within locus coeruleus is unique in its utilization of a defined control group, experimental groups consisting of strictly defined replications, accurate three-dimensional reconstruction, and statistical comparisons. The demonstrated spatial heterogeneity of locus coeruleus neurons with respect to efferent projections can now be compared to the spatial distributions of other cellular characteristics such as soma morphology, colocalized transmitters and physiological properties. Presumably, such spatial segregation reflects the operation of functionally important organizing principles within the nucleus.

Animals↗
PubMed1992

Origin of the medial ulnar collateral ligament.

The anatomic features of the origin of the anterior medial collateral ligament of the elbow were studied in 10 cadaver elbows to determine the percentage of the medial epicondyle that can be removed without violating the ligament, and whether or not this ligament attaches to the condyle as well as to the epicondyle. In all specimens the anterior medial collateral ligament originated exclusively from the anteroinferior surface of medial epicondyle and had no attachment to the condyle. Only 20% of the width of the medial epicondyle in the coronal plane can be removed without violating a portion of the origin of the anterior medial collateral ligament, an essential stabilizer of the elbow. Excision of the entire epicondyle for ulnar neuropathy would completely detach this ligament from its origin and might therefore potentiate instability. Since the ligament originates on the anteroinferior surface of the epicondyle, more bone can be removed with less violation of the anterior medial collateral ligament origin if the plane of the osteotomy lies between the sagittal and coronal planes.

Cadaver↗
PubMed2005

Subpallial origin of part of the calbindin-positive neurons of the claustral complex and piriform cortex.

The aim of the present study was to investigate whether part of the calbindin-positive neurons of the claustral complex and piriform cortex originate in the subpallium. To that end, we prepared organotypic cultures of embryonic telencephalic slices, and applied the cell tracker CMTMR to the ventricular/subventricular zone of the lateral or medial ganglionic eminence. Following 48 h of incubation, we observed a number of CMTMR-labeled cells (showing red fluorescence) of subpallial origin in the claustral complex and piriform cortex. To know whether some of these cells of subpallial origin were calbindin-positive, we performed immunofluorescence for calbindin using an Alexa 488-conjugated secondary antiserum (green fluorescence). Our results showed that some of the CMTMR-labeled cells of subpallial origin in the claustral complex and piriform cortex are calbindin-positive (and possibly GABAergic). The subpallial origin of part of these cells was confirmed by observation of double labeled neurons in the claustral complex that expressed both Lhx6 mRNA (a marker of cells derived from the medial ganglionic eminence) and calbindin. Future studies will be required to analyze the existence of a subpopulation of non-GABAergic calbindin cells in the claustral complex and piriform cortex, and to know their origin.

Animals↗
PubMedDate not supplied

Intracardiac fibroblasts, but not bone marrow derived cells, are the origin of myofibroblasts in myocardial infarct repair.

UNLABELLED: Origin of myofibroblasts in infarcted myocardium was examined by using rats in which bone marrow of green fluorescent protein (GFP)-transgenic mice had been transplanted. GFP was not detected in myofibroblasts at either 3 or 7 days after infarction, suggesting that proliferating myofibroblasts in infarcted myocardium are derived from resident fibroblasts rather than circulating precursor cells of bone marrow origin. BACKGROUND: Myofibroblasts play important roles in the repair process of myocardial infarct, and their origin has been assumed to be interstitial fibroblasts in the heart. However, bone marrow-derived myofibroblasts have recently been identified in pathological fibrosis in extracardiac tissues. In this study, we aimed to determine whether some of the myofibroblasts in infarcted myocardium are derived from circulating precursor cells of bone marrow origin. METHODS AND RESULTS: Bone marrow (BM) of GFP-transgenic mice was transplanted into nude rats, and their coronary arteries were occluded for 60 min and reperfused for 3 or 7 days. Non-BM-transplanted rats served as controls. At 3 days after infarction, some endothelial cells were GFP-positive, indicating that they were of bone marrow origin. Predominant cells in infarcted regions were macrophages and neutrophils, and there were only a small number of vimentin-positive cells and fewer myofibroblasts, both of which were GFP-negative. At 7 days after infarction, there were numerous myofibroblasts in granulation tissue replacing necrotic myocytes, and none of them showed GFP signals, whereas some cells were positive for both GFP and vimentin. Appearance of myofibroblasts and extent of the infarct repair in BM-transplanted and those in non-transplanted rats were similar. CONCLUSIONS: The findings in this study suggest that proliferating myofibroblasts in infarcted myocardium are derived from resident fibroblasts rather than circulating precursor cells of bone marrow origin.

Animals↗
PubMed2004

Presence of drug resistance in intestinal lactobacilli of dairy and human origin in Turkey.

The prevalence of different resistance genes was investigated in lactobacilli of human and dairy origin by PCR. The presence of erm, van, tet, and cat-TC genes were determined in 16 raw milk, 15 cream, 10 yogurt, 50 hand-made cheese, and 20 industrially produced white-cheese samples of dairy origin and 16 mouth, 32 fecal, and 36 vaginal samples from different subjects of human origin. Lactobacilli of dairy and human origin were found to carry only erm(B) and tet(M) genes. The majority of the isolates, Lactobacillus crispatus (61), Lactobacillus gasseri (49), Lactobacillus plantarum (80) studied were found to harbor either erm(B) or tet(M) gene or both. No resistant lactobacilli was found in raw-milk and cream samples. All the human fecal samples and the majority of vaginal (29 of 36) and mouth (10 of 14) samples were found to carry the resistance genes. While a third of the hand-made cheeses carried resistant lactobacilli only one industrially produced cheese was found to carry resistant lactobacilli. Furthermore, the genes were found in the non-starter species, Lactobacillus acidophilus and Lb. plantarum, indicating that industrially produced cheeses in this respect could be considered more favorable. These results indicate that drug resistance seems to be very common in Turkey. Even though the number of dairy samples harboring the resistance genes (17 of 111) is smaller in regards to human samples, 10% of them were still found to carry the resistance genes as well. The presence of the resistance genes in majority of the samples of human origin and in minority of the samples of dairy origin indicates that drug resistance may be acquired in the intestinal tract during passage and spread to dairy products by the hands of workers during production.

Animals↗
PubMed2006

Mitochondrial molecular clocks and the origin of the major Otocephalan clades (Pisces: Teleostei): A new insight.

The Otocephala, a clade including ostariophysan and clupeomorph teleosts, represents about a quarter of total fish species diversity, with about 1000 genera and more than 7000 species. A series of recent papers have defended that the origin of this clade and of its major groups may be significantly older than the oldest fossils of each of these groups suggest. Some of these recent papers explicitly defend a Pangean origin for some otocephalan groups such as the Siluriformes or Cypriniformes. To know whether or not the otocephalans as a whole, and particularly the mainly freshwater, cosmopolitan otophysans could have originated before the splitting of the Pangean supercontinent is of extreme importance, since otophysan fishes are among the most useful animal groups for the determination of historical continental relationships. In the present work we examined divergence times for each major otocephalan group by an analysis of complete mtDNA sequences, in order to investigate if these divergence times support the hypotheses advanced in recent studies. The complete mtDNA sequences of nine representative non-otocephalan fish species and of twenty-one representative otocephalan species was compared. The present study is thus, among the studies dealing with molecular divergence times of teleosts, the one in which a greater number of otocephalan species are included. The divergence times obtained support that the major otocephalan groups had a much older origin than the oldest fossil records available for these groups suggest. The origin of the Otocephala is estimated as having occurred about 282 Mya, with the origin of the Otophysi being estimated at about 251 Mya.

Animals↗