Origins and termini of adenovirus type 2 DNA replication.
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Adenoviridae↗
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Results for “Origin cell mapping”
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A new alpha-particle irradiator with absolute dosimetric determination.
A new experimental setup for uniform alpha-particle irradiation of cells in vitro is described. The alpha-particle irradiator is based on a radioactive (212)Pb/(212)Bi source. In the experimental setup proposed, cells are grown directly on a polylysine-coated track-etch material that forms the base of custom-made cell dishes. Alpha-particle irradiation is done through the base of the dish. Immediately prior to irradiation, the cell dish is scanned under a microscope, and images of cells with the corresponding coordinates are saved. After irradiation and after the biological end point under study has been determined, the cell dish is etched to develop alpha-particle tracks in the dish base. A microscope image series of alpha-particle track images is obtained by accurately revisiting every original (preirradiation) cell position in the track-etched dish. The number of alpha-particle traversals of each individual cell is scored by mapping images of alpha-particle tracks onto the images of cells recorded prior to irradiation. The uncertainty of the alpha-particle hit determination is 0.9 microm. The procedure described thus presents a method for radiobiological experiments with absolute, rather than statistical, cell dosimetry.
Air↗
Genetic rearrangements of plasmids containing mtDNA inserts: possible poison sequence.
Restriction mapping of recombinant plasmids indicated the presence of poison sequence(s) in monkey mtDNA. These plasmids were constructed from a 5.2 K.b. BglII mtDNA fragment and pRSVneo or pdel9 as cloning vectors. The poison sequence(s) caused genetic rearrangement of the vectors' nucleotide sequences. Deletion of the suspected poison sequence(s) from the mtDNA fragment increased the transformation efficiency of the produced recombinant plasmids and conserved the vectors' original nucleotide sequences.
Animals↗
Is Crohn's disease caused by a mycobacterium? Comparisons with leprosy, tuberculosis, and Johne's disease.
Although Crohn's disease is considered to be autoimmune in origin, there is increasing evidence that it may have an infectious cause. The most plausible candidate is Mycobacterium avium subspecies paratuberculosis (MAP). Intriguingly, Koch's postulates may have been fulfilled for MAP and Crohn's disease, even though they still have not been met for Mycobacterium leprae and leprosy. In animals MAP causes Johne's disease, a chronic wasting intestinal diarrhoeal disease evocative of Crohn's disease. Johne's disease occurs in wild and domesticated animals, including dairy herds. Viable MAP is found in human and cow milk, and is not reliably killed by standard pasteurisation. MAP is ubiquitous in the environment including in potable water. Since cell-wall-deficient MAP usually cannot be identified by Ziehl-Neelsen staining, identification of MAP in human beings requires culture or detection of MAP DNA or RNA. If infectious in origin, Crohn's disease should be curable with appropriate antibiotics. Many studies that argue against a causative role for MAP in Crohn's disease have used antibiotics that are inactive against MAP. However, trials that include macrolide antibiotics indicate that a cure for Crohn's disease is possible. The necessary length of therapy remains to be determined. Mycobacterial diseases have protean clinical manifestations, as does Crohn's disease. The necessity of stratifying Crohn's disease into two clinical manifestations (perforating and non-perforating) when interpreting the results of antibiotic therapy is discussed. Rational studies to evaluate appropriate therapies to cure Crohn's disease are proposed.
Animals↗
Transcription of the chicken alpha 2 (Type I) collagen gene by homologous cell-free extracts.
We have used two methods to detect specific transcription of the chicken alpha 2 (type I) collagen gene in cell-free extracts derived from Rous sarcoma virus-transformed chicken embryo fibroblasts. The first method is a modification of the S1 nuclease mapping procedure which utilizes a DNA probe labeled with 32P at the 5' end of the HindIII linker originally used to clone the collagen promoter region into PBR322. The probe distinguishes newly made, specific RNA from endogenous RNA and nonspecific transcripts. Using this procedure we have found that chicken whole cell extracts support accurate initiation of transcription of the chicken alpha 2 (type I) collagen DNA template. Addition of either creatine phosphate, GTP, or UTP to concentrations of approximately 3 to 5 mM was found to stimulate RNA polymerase II transcription by 5- to 10-fold. The second method employs an avian myeloblastosis virus reverse transcriptase-catalyzed primary extension procedure, rendered in vitro-specific by use of a pBR322 fragment as primer. These two techniques should be useful for analyzing specific transcription in other types of cell-free extracts.
Animals↗
Monoclonal antibodies to the major nonstructural nuclear protein of minute virus of mice.
Monoclonal antibodies were raised against a bacterial fusion protein containing amino acids 364 to 623 of the major nonstructural protein, NS-1, of minute virus of mice (MVMp), an autonomous parvovirus. By immunoblot analyses, these antibodies all recognized an 83-kDa protein in MVM-infected mouse fibroblast cells. Indirect immunofluorescence studies showed that five of the six react against a nuclear protein in MVM-infected mouse cells resulting in discrete foci of fluorescence. These foci do not correspond with the nucleoli, the site of MVM DNA replication. The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen and showed that four distinct epitopes were recognized by the different antibodies. A 25-amino-acid peptide was used in competition ELISAs to confirm the location of the epitope recognized by two antibodies CE10 and AC6. Preliminary characterization of an NS-1/NS-2 fusion protein synthesized in insect cells using a baculovirus expression vector showed that this fusion protein is also localized within the nucleus; however, in contrast, the full-length NS-1 polypeptide is located within the cytoplasm.
Amino Acid Sequence↗
Partitioning of plasmid R1 in Escherichia coli. I. Kinetics of loss of plasmid derivatives deleted of the par region.
The stability of inheritance of plasmid R1drd-19 was tested. The copy number of the plasmid was determined in two different ways: As the ratio between covalently closed circular DNA and chromosomal DNA, and by quantitative determination of single-cell resistance to ampicillin. In the latter case, strains carrying the R1 ampicillin transposon Tn3 on prophage lambda was used as standard. The values were transformed to copy number per cell by using the Cooper-Helmstetter model for chromosome replication as well as by determination of chromosomal DNA per cell by the diphenylamine method. The copy number was found to be five to six per cell (or about four per newborn cell). Nevertheless, plasmid R1drd-19 was found to be completely stably inherited. This stability was shown not to be due to retransfer of the plasmid by the R1 conjugation system, since transfer-negative derivatives of the plasmid were also completely stably inherited. Smaller derivatives of plasmid R1drd-19 were found to be lost at a frequency of about 1.5% per cell generation. The copy-number control was not affected in these miniplasmids, since their copy numbers were the same as that of the full size plasmid. Quantitatively, the instability of the miniplasmids was in accord with random partitioning. It is, therefore, suggested that the plasmid R1drd-19 carries genetic information for partitioning (par) of plasmid copies at cell division, and that the par mechanism is distinct from the copy number control (cop) system. Finally, the par gene maps on the resistance transfer part of the plasmid, but far away from the origin of replication and the so-called basic replicon; this is in accord with the approximate location of the repB gene (Yoshikawa, 1974, J. Bacteriol., 118, 1123-1131).
Cell Division↗
Traction forces exerted through N-cadherin contacts.
BACKGROUND INFORMATION: Mechanical forces play an important role in the organization, growth and function of living tissues. The ability of cells to transduce mechanical signals is governed by two types of microscale structures: focal adhesions, which link cells to the extracellular matrix, and adherens junctions, which link adjacent cells through cadherins. Although many studies have examined forces induced by focal adhesions, there is little known about the role of adherens junctions in force-regulation processes. The present study focuses on the determination of force transduction through cadherins at a single cell level. RESULTS: We characterized for the first time the distribution of forces developed by the cell through cadherin contacts. A N-cadherin (neural cadherin)-Fc chimaera, which mimicks the cell adhesion molecule N-cadherin, was immobilized on a muFSA (micro-force sensor array), comprising a dense array of vertical elastomer pillars, which were used both as a cell culture support for N-cadherin-expressing C2 myogenic cells and as detectors for force mapping. We coated the top of the pillars on which cells adhere and recruit adhesion complexes and F-actin. Individual pillar bending allowed the measurement of forces that mainly developed at the cell edge and directed toward their centre. Similar force distribution and amplitude were detected with an unrelated cell line of neuronal origin. Further comparison with forces applied by cells on pillars coated with fibronectin indicates that mechanical stresses transduced through both types of adhesions were comparable in distribution, orientation and amplitude. CONCLUSIONS: These results present a versatile method to measure and map forces exerted by cell-cell adhesion complexes. They show that cells transduce mechanical stress through cadherin contacts which are of the same order as magnitude of those previously characterized for focal adhesions. Altogether, they emphasize the mechanotransduction role of cytoskeleton-linked adhesion receptors of the cadherin family in tissue cohesion and reshaping.
Actins↗
Construction, analysis, and application of a radiation hybrid mapping panel surrounding the mouse agouti locus.
The region surrounding the agouti coat color locus on mouse Chromosome 2 contains several genes required for peri-implantation development, limb morphogenesis, and segmentation of the nervous system. We have applied radiation hybrid mapping, a somatic cell genetic technique for constructing long-range maps of mammalian chromosomes, to eight molecular markers in this region. Using a mathematical model to estimate the frequency of radiation-induced breakage, we have constructed a map that spans approximately 20 recombination units and 475 centirays8000. The predicted order of markers, Prn-p-Pygb-Emv-13-Psp-Xmv-10-Emv-15-Src-Ada, is consistent with a previously derived multipoint meiotic map for six of the eight markers and suggests that Xmv-10 may lie relatively close to one or more of the agouti recessive lethal mutations. The resolution of our map is approximately 40-fold higher than the meiotic map, but the median retention frequency of mouse DNA in hybrid cells, 0.12, is 4-fold lower than similar experiments with human chromosomes. From one of the radiation hybrid lines that contained a minimum amount of mouse DNA, 25 independent cosmids were isolated with a mouse-specific hybridization probe. Single-copy fragments from two of these cosmids were shown to originate from mouse Chromosome 2, and the meiotic map position of one was found to be within 10 recombination units of the region of interest. Our results indicate more precise map positions for Pygb and Xmv-10, demonstrate that radiation hybrid mapping can provide high-resolution map information for the mouse genome, and establish a new method for isolating large fragments of DNA from a specific subchromosomal region.
Animals↗
Mapping of the human homologue (ORC1L) of the yeast origin recognition complex subunit 1 gene to chromosome band 1p32.
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Animals↗
Conserved chromatin structure in c-myc 5'flanking DNA after viral transduction.
The role of local sequence information in establishing the chromatin structure of the human c-myc upstream region (MUR) was investigated. Adeno-associated virus (AAV)-mediated gene transduction was used to introduce an additional unrearranged copy of the 2.4 kb HindIII-XhoI fragment of the MUR into a novel location in the genome in each of two cloned HeLa cell lines. The AAV-based rep- cap- viral vector SKMA used to transduce the MUR retained only 1.4 kb (24%) of the AAV genome and could accommodate inserts as large as 2.4 kb. SKMA was capable of infecting HeLa cells and integrating into the host genome at single copy number. Integration may have occurred at a preferred site in the HeLa genome, but this site was apparently distinct from the previously identified preferred AAV integration site on human chromosome 19. Indirect end-labelling was used to map DNase I and micrococcal nuclease (MNase) cleavage sites over the transduced c-myc sequences and the endogenous c-myc loci in infected HeLa cells. A similarly ordered chromatin domain, extending 5' from c-myc promoter P0, was found to exist at the transduced c-myc locus in each clone. The position and relative sensitivity of 13 MNase cleavage sites and five DNase I hypersensitive sites, originally identified at the endogenous MUR in non-transduced cells, were shown to be conserved when this DNA was moved to a new chromosome site. A conserved DNase I hypersensitive site also was mapped to the region between the left AAV terminal repeat and AAV promoter P5. These results suggest that the information required to establish the particular chromatin structure of the MUR resides within the local DNA sequence of that region.
Adenoviruses, Human↗
Glomerulus-specific synchronization of mitral cells in the olfactory bulb.
Odor elicits a well-organized pattern of glomerular activation in the olfactory bulb. However, the mechanisms by which this spatial map is transformed into an odor code remain unclear. We examined this question in rat olfactory bulb slices in recordings from output mitral cells. Electrical stimulation of incoming afferents elicited slow ( approximately 2 Hz) oscillations that originated in glomeruli and were highly synchronized for mitral cells projecting to the same glomerulus. Cyclical depolarizations were generated by glutamate activation of dendritic autoreceptors, while the slow frequency was determined primarily by the duration of regenerative glutamate release. Patterned stimuli elicited stimulus-entrained oscillations that amplified weak and variable inputs. We suggest that these oscillations maintain the fidelity of the spatial map by ensuring that all mitral cells within a glomerulus-specific network respond to odor as a functional unit.
2-Amino-5-phosphonovalerate↗
Location of cells of origin of spinothalamic tract in lumbar enlargement of the monkey.
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Action Potentials↗
Proteomic analysis of nasal cells from cystic fibrosis patients and non-cystic fibrosis control individuals: search for novel biomarkers of cystic fibrosis lung disease.
Potential biological markers for cystic fibrosis (CF) lung disease were identified by comparative proteomics profiling of nasal cells from deletion of phenylalanine residue 508 (F508del)-homozygous CF patients and non-CF controls. From the non-CF 2-DE gels, 65 spots were identified by MS, and a reference 2-DE map was thus established. The majority of those correspond to ubiquitously expressed proteins. Consistent with the epithelial origin of this tissue, some of the identified proteins are epithelial markers (e.g. cytokeratins, palate lung and nasal epithelium clone protein (PLUNC), and squamous cell carcinoma antigen 1). Comparison of this protein profile with the one similarly obtained for CF nasal cells revealed a set of differentially expressed proteins. These included proteins related to chronic inflammation and some others involved in oxidative stress injury. Alterations were also observed in the levels of cytoskeleton proteins, being probably implicated with cytoskeleton organization changes described to occur in CF-airways. Lower levels were found for some mitochondrial proteins suggesting an altered mitochondrial metabolism in CF. Differential expression was also found for two more enzymes that have not been previously associated to CF. Further studies will clarify the involvement of such proteins in CF pathophysiology and whether they are targets for CF therapy.
Adolescent↗
Mapping replication origins, pause sites, and termini by neutral/alkaline two-dimensional gel electrophoresis.
Neutral/alkaline two-dimensional gel electrophoresis is a robust, easily interpretable, sensitive technique that has yielded insights about the in vivo replication of many types of DNA, from multicopy yeast plasmids to single-copy chromosome regions in unsynchronized mammalian cells. It can provide information about directions of replication fork movement and locations of replication origins, termini, and pause sites. Especially when combined with its partner technique, neutral/neutral two-dimensional gel electrophoresis, it is a method of choice for investigation of unknown situations.
Animals↗
[Toward a new definition of malignant histiocytosis in children].
The anaplastic large cell, CD30 positive lymphomas represent a heterogeneous group of lymphomas in which immunocytochemical and molecular investigations have demonstrated the existence of malignancies of T, B or undetermined origin. The recent identification, in a few cases of this group of a chromosomal 5q35 breakpoint may allow the individualization of a peculiar disease entity. In these cases, the 5q35bp has been found to be a permanent abnormality, present in five human permanent cell lines and associated with various translocations including t(2;5), t(1;5) and t(5;6). A primitive myelo-monocytic origin of these 5q35bp cell is suggested on the basis of the following arguments: they express the c-fms proto-oncogene which encodes the macrophage growth receptor (CSF-1-R) the c-fms is mapped on 5q33,34 close to the 5q35bp the express spontaneously CD68 the treatment by phorbol-diester of a 5q35bp cell line (DEL cell line) induces an immunodependent phagocytosis and a modulation of expression of c-fms, CSF-1 and TNF alpha. Because some 5q35bp cell lines also presents rearrangements for TCR-bêta, or IgF, these data suggest an ancestral stem cell origin, prior to the T, B and myelomonocytic differentiation. Whatever its origin, the 5q35bp abnormality has been mainly encountered in children's malignancies. It has been constantly associated with clinical and biological manifestations of a condition recognized by paediatricians as malignant histiocytosis. For this reason, the 5q35bp may today represent the best criterion for the identification of malignant histiocytosis in childhood.
Child↗
Genomic characterization of human HMGIC, a member of the accessory transcription factor family found at translocation breakpoints in lipomas.
HMGIC, a member of the HMGI family of high mobility group proteins, is disrupted in 3/3 lipomas characterized by 12q14-q15 rearrangements. To define the genomic structure of the HMGIC gene, YACs from the HMGIC locus were identified and subcloned in lambda FIXII, and genomic lambda clones containing the HMGIC exons were isolated. The HMGIC gene consists of five exons that span at least 60 kb and encodes a 4.1-kb transcript. The coding region is 330 bp, the 5'UTR is 854 bp, and the 3'UTR is 2966 bp. Intron 3, which separates the DNA-binding domains from the acidic domain, is unusually large ( > 25 kb) and is the site of disruption in lipomas with 12q14-q15 translocations. The genomic organization of HMGIC should further our understanding of the 12q14-q15 region, where other neoplasms of mesenchymal origin are also mapped.
Animals↗
Retrograde and anterograde axonal transport demonstrated by intracerebral injection of a labeled protein-acylating agent.
Following injection of the tritium-labeled protein-acylating agent, N-succinimidyl propionate, into the neostriatum in rats, retrograde labeling of nigrostriatal cells in the pars compacta of the subtantia nigra and anterograde labeling of the striatonigral terminal field in the nigral pars reticulata were demonstrated by autoradiography, at 18 hr survival. Labeling of the corticopontine terminal field and of corticothalamic cell bodies in cortical layer 6 were also seen, deriving from corticofugal fibers of passage through the striatal injection site. In comparison, neostriatal injection of horseradish peroxidase was found to yield nigral cell and terminal field labeling, but the corticopontine terminal field was not labeled, and very few cortical cells in layer 6 were labeled. These results show that the retrograde and anterograde axonal transport of endogenous proteins can be profitably studied by this in vivo protein-acylating technique, and suggest that the method may prove useful as a connection-tracing technique, especially because of the novel attribute of labeling terminal fields by anterograde axonal transport following injections into white matter axonal bundles, with concomitant labeling od the cell bodies of origin of these axons.
Animals↗