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At least 433 records · Page 24Linked to original sources
PubMed2006

Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: Changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes.

We examined the effects of genetic transformation by Agrobacterium rhizogenes on the production of tylophorine, a phenanthroindolizidine alkaloid, in the Indian medicinal plant, Tylophora indica. Transformed roots induced by the bacterium grew in axenic culture and produced shoots or embryogenic calli in the absence of hormone treatments. However, hormonal treatment was required to regenerate shoots in root explants of wild type control plants. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes, which include, short internodes, small and wrinkled leaves, more branches and numerous plagiotropic roots. Plants regenerated from transformed roots showed increased biomass accumulation (350-510% in the roots and 200-320% in the whole plants) and augmented tylophorine content (20-60%) in the shoots, resulting in a 160-280% increase in tylophorine production in different clones grown in vitro.

Alkaloids↗
PubMed1985

Focal contacts of normal and RSV-transformed quail cells. Hypothesis of the transformation-induced deficient maturation of focal contacts.

Morphology and distribution of cell-substrate contacts and their association with microfilament bundles in normal and RSV-transformed quail fibroblasts (16Q line) were studied by indirect immunofluorescence. The focal contacts were visualized by antibody exclusion method using monoclonal antibody to 80 kD serum protein adsorbed on the substratum. Embryo quail cells formed focal contacts of two morphological types: (1) small punctate; and (2) elongated large contacts. These two variants of contacts were designated respectively as dot and dash contacts. Both of focal contacts contained vinculin and alpha-actinin. Double immunofluorescence staining with polyclonal antibody to actin and monoclonal antibody to vinculin revealed that the dot contacts, in contrast to the dash ones, were not associated with microfilament bundles. The dot contacts were localized mostly near the active cell edges, while the dash contacts were found near the retracted cell edges and also under the central parts of the cell. We suppose that dot contacts represent initial structures which then can undergo maturation transforming them into dash contacts. RSV-transformed 16Q cells had predominantly the dot contacts which were not only located at the edges but also in the more central parts of the lamella. The dash contacts were present only in the minority of 16Q cells. RSV transformation is assumed to affect not the ability of cells to form initial dot contacts, but the maturation of dot contacts into dash contacts.

Actins↗
PubMed1991

v-myb transformation of Xeroderma pigmentosum human fibroblasts: overexpression of the c-Ha-ras oncogene in the transformed cells.

Human Xeroderma pigmentosum "normal" fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental "normal" AS16 cells and a revertant clone (ASKXA Cl 1.1 G). Our results lead to the conclusion that the XP fibroblasts are phenotypically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.

Animals↗
PubMed1985

Transformation by polyoma ts-a mutants. I. Characterization of the transformed phenotype.

Seven clonal lines of Fischer rat cells transformed with ts-a mutants of polyoma virus were studied. Four clones are characterized by a temperature-sensitive (ts) and three clones by a temperature-insensitive-transformed phenotype. Six clones have retained a functional though temperature-sensitive large T antigen, as judged by a 10- to 20-fold amplification of viral sequences in clones grown at low temperature compared to those grown at high temperature. No amplification is observed in one non-ts clone. As analyzed by Southern blotting, no obvious difference appears in the integration pattern of viral sequences in ts and non-ts clones concerning the number of sites of genome integration, the presence or absence of tandem repeats of the viral genome, or the absence of specific viral sequences. In autoradiograms of gel-electrophoresed immunoprecipitates, no correlation can be drawn between the amounts of either large T antigen or middle T antigen and the type of transformed state of the clones under the conditions tested. In assays of the middle T-antigen-associated kinase, no reproducible difference can be observed between the non-ts and ts clones. Finally, no correlation was observed between a temperature-insensitive phenotype and the production of an N-terminal fragment of large T antigen. Thus the molecular basis for the difference between ts-a transformants with ts or non-ts phenotypes remains elusive.

Antigens, Viral, Tumor↗
PubMed1986

Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation.

Mutagenesis of glycine 2 of p60src, the transforming protein of Rous sarcoma virus (RSV), yields a protein that is neither myristylated nor bound to cellular membranes. Although these mutant viruses retain full tyrosine protein kinase activity, they are transformation-defective. We examined in detail tyrosine phosphorylation of cellular polypeptides and the phenotype induced by infection with two such viruses. Infection failed to cause growth in agar, cytoskeletal reorganization, or changes in fibronectin synthesis and protease secretion. Strikingly, tyrosine phosphorylation of the known substrates of p60src was extensive, and differed from that found in wild-type transformed cells only quantitatively. There was no apparent correlation between the extent to which any of eight known protein substrates of p60src were phosphorylated and the phenotype of infected cells. We suggest that the phosphorylation of as yet unidentified proteins, which are probably found in cellular membranes, is essential for transformation by RSV.

Actins↗
PubMed1986

A gel-electrophoretic analysis of protein ADP-ribosylation in polyoma virus-transformed and non-transformed BHK-21/C13 fibroblasts.

We have examined a variety of conditions for solubilizing and electrophoresing cell proteins in order to define optimum conditions for studying proteins modified by ADP-ribosylation. We have identified conditions in which proteins can be quantitatively extracted from cells in an undegraded form with the protein-ADPribose linkages intact. Effective measures include boiling cells briefly (4 min) in the presence of 2% SDS and 2 M urea at pH 6.8. Both SDS and urea were present in the 6-18% gradient polyacrylamide gel matrix used for electrophoresis. Under these conditions good resolution of proteins of a wide molecular-weight range is obtained. This system has been used to compare protein ADP-ribosylation in non-transformed and polyma virus-transformed baby hamster kidney (BHK) fibroblasts, since the latter cells have a greater NAD+ ADP-ribosyltransferase activity (measured in isolated nuclei and permeabilized cells). Addition of DNAase to permeabilized BHK cells over the range 10-150 micrograms led to a progressively greater activation of transferase compared with controls. When PyY cells were used, however, maximum activation was achieved with only 10 micrograms of DNAase, further additions producing a successively smaller activation relative to control cells without added nuclease. There were also differences between these cells in response to salt. Addition of NaCl (to about 0.3 M) to BHK cells resulted in various extents of transferase activation, whereas any addition of NaCl to the incubate of permeabilized PyY cells decreased transferase activity. These different enzyme activities between this transformed and non-transformed cell line are for the most part not reflected in the protein modification profiles seen on autoradiograms of acrylamide gels after electrophoresis 32P-labelled proteins. A variety of proteins are modified and their molecular weights depend on the NA concentration in the permeabilized cell incubation. At 0.5 microM NAD+ there were two major acceptors with Mr values of 14 kDa and 30 kDa, and at 100 microM NAD+, three major acceptors, with Mr values of 19 kDa. 45 kDa and greater than 170 kDa. NAD concentrations of between 1 microM and 100 microM had no further effect on protein ADP-ribosylation profiles, except for the protein(s) of Mr greater than 170 kDa, pointing to a critical difference around 0.5-1.0 microM substrate. In some experiments, however, a difference was observed in the intensity of radioactivity in two bands. This may represent two different proteins, or a single protein modified to different extents.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1985

Spreading of non-transformed and transformed cells.

Mechanisms of cellular reactions responsible for the spreading of non-transformed cultured tissue cells on the surface of various substrata and relationships of these reactions to the control of cell proliferation are reviewed; the special role of the membrane-cytoskeleton interactions leading to extension and attachment of pseudopods is stressed. Transition of cells from non-transformed to transformed phenotype is characterized by decreased spreading and by decreased dependence of proliferation on spreading. Manifestations of both of these spreading-associated changes are reviewed and their possible mechanisms are discussed. It is suggested that cell transition to transformed phenotype involves shift of an equilibrium between the reactions induced by the two groups of membrane-bound ligands: those attached and those not attached to the substratum.

Animals↗
PubMed1984

Molecular phenotyping of non-transformed and chemically transformed C3H10T1/2 mouse fibroblasts.

A systematic molecular phenotyping approach based on two-dimensional gel electrophoresis is being applied in an attempt to identify protein changes associated with malignant transformation. Using the C3H10T1/2 mouse cell line, two-dimensional polypeptide maps of the non-transformed cell line, several chemically transformed lines and a tumour cell line were compared. Although there is a large degree of similarity between the protein profiles of all cell lines, clear differences are evident. Initial results are consistent with the view that many of the protein changes are incidental to malignant transformation. Changes induced by 3-methylcholanthrene are retained after transplantation of the cells into nude mice.

Animals↗
PubMed1985

Effects of factors derived from a tumor clonal cell line on DNA synthesis of transformed and non transformed cells.

Conditioned medium from neoplastic thyroid cell cultures, extracts of tumors developed by identical cells in isogeneic Fisher 344 rats and serum from those tumor-bearing animals, were tested in pulse thymidine labelled experiments on a transformed and two non transformed cell lines. Tumor extract and conditioned medium inhibited DNA synthesis. Tumor-bearing rat serum increased DNA synthesis in a cerebellar transformed cell line, but no in chick embryo fibroblasts or in aorta non transformed cells.

Animals↗
PubMed1977

Transformation with specific fragments of adenovirus DNAs. II. Analysis of the viral DNA sequences present in cells transformed with a 7% fragment of adenovirus 5 DNA.

Five clones of rat kidney cells transformed by a small restriction endonuclease fragment of adenovirus 5 (Ad5) DNA (fragment HsuI G, which represents the left terminal 7% of the adenovirus genome) were analyzed with respect to the viral DNA sequences present in the cellular DNAs. In these analyses, the kinetics of renaturation of 32P-labeled specific fragments of Ad5 DNA was measured in the presence of a large amount of DNA extracted either from each of the transformed cell lines or from untransformed cells. The fragments were produced by digestion of 32P-labeled adenovirus 5 DNA with endo R.HsuI, or by digestion of 32P-labeled fragment HsuI G of adeno 5 DNA with endo R.HpaI. All five transformed lines were found to contain DNA sequences homologous to 75--80% of Ad5 fragment HsuI G only. Clones II and V contained approximately 48 copies per quantity of diploid cell DNA, clone VI about 35 copies, clone IV 22 copies and clone III 5--10 copies. These results indicate that a viral DNA segment as small as 5.5% of the Ad5 genome, contains sufficient information for the maintenance of transformation.

Adenoviruses, Human↗
PubMed1993

Genetic manipulation of Campylobacter: evaluation of natural transformation and electro-transformation.

Two methods, natural transformation and electro-transformation, for the introduction of DNA into nine strains of Campylobacter jejuni were compared. Both methods were successful with a limited number of strains. Natural transformation was efficient only for the introduction of C. jejuni chromosomal DNA, while electro-transformation was also applicable for the introduction of Escherichia coli-derived vector DNA into at least one C. jejuni strain. The efficiency of DNA recombination after entry was determined using C. jejuni chromosomal DNA containing disrupted flagellin genes of C. jejuni or suicide vectors containing a portion of these genes. In the latter case, DNA recombination occurred with as little as 200-bp homology present, indicating that only short homologous DNA segments are required.

Campylobacter jejuni↗
PubMed2000

Anti-transforming nature of ascorbic acid and its derivatives examined by two-stage cell transformation using BALB/c 3T3 cells.

The anti-transforming effects of sodium ascorbate and its stable derivatives were examined in the two-stage transformation assay. When BALB/c 3T3 cells were treated with 0.2 microg/ml 20-methylcholanthrene as an initiator, and 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate as a promoter, the addition at the promotion stage of L-ascorbic acid-2-phosphate ester magnesium (APM) was most marked in the inhibition of transformation. The inhibitory effects of sodium ascorbate and ascorbic acid-2-glucoside (AG) were comparable, but weaker than those of APM; L (+)-ascorbic acid-2-sulfate ester disodium 2H(2)O showed little effect. When phorbol 12, 13-didecanoate or tumor necrosis factor alpha (TNF-alpha) were used as promoters, APM also effectively suppressed transformation.

3T3 Cells↗
PubMed2000

The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation.

This paper describes a so-called ternary transformation system for plant cells. We demonstrate that Agrobacterium tumefaciens strain LBA4404 supplemented with a constitutive virG mutant gene (virGN54D) on a compatible plasmid is capable of very efficient T-DNA transfer to a diverse range of plant species. For the plant species Catharanthus roseus it is shown that increased T-DNA transfer results in increased stable transformation frequencies. Analysis of stably transformed C. roseus cell lines showed that, although the T-DNA transfer frequency is greatly enhanced by addition of virGN54D, only one or a few T-DNA copies are stably integrated into the plant genome. Thus, high transformation frequencies of different plant species can be achieved by introduction of a ternary plasmid carrying a constitutive virG mutant into existing A. tumefaciens strains in combination with standard binary vectors.

Acetophenones↗
PubMed1976

Chemical transformation of Chinese hamster cells: II. Appearance of marker chromosomes in transformed cells.

The chromosomes of 12 samples of cultured Chinese hamster kidney and prostate cells (4 normal and 8 transformed), whose tissue culture properties have already been described (Kirkland, 1976) have been examined for numerical change and for the appearance of abnormal markers. Six transformed kidney subclones contained a consistent telocentric marker not present in the normal parental cell, and Giemsa banding demonstrated this to be the centromere and the long (q) arm of the number 4 chromosome in all cases. Two transformed prostate subclones also contained a consistent telocentric marker, not present in similarly derived normal subclones or in the normal parental cell, and Giemsa banding demonstrated this to be a different fragment (the centromere and most of the p arm) of the number 4 chromosome. It is believed that the use of a mixed-serum culture medium, designed to stabilize the karyotype of cultured Chinese hamster cells, is at least partly responsible for the detection of these transformation-associated chromosome changes.

Carcinogens↗
PubMed1970

DNA-induced transformation in Drosophila: locus-specificity and the establishment of transformed stocks.

The genetic changes induced in Drosophila melanogaster by DNA obtained from flies differing at specified genetic loci exhibit the general features of transformation, namely locus specificity and heritability. These properties have provided the basis for the establishment of transformed stocks. The phenomenon also exhibits striking special features, prominent among which is the observation that transformed individuals are always mosaics and that whole-body transformants are absent even in established stocks. The exosome model (which proposes that DNA segments enter the cells of treated individuals and become firmly associated with their homologous chromosome segments, but are never integrated into the linear structure of the chromosome) is formulated to fit these observations.

Animals↗
PubMed1972

Evidence for conversion of heteroduplex transforming DNAs to homoduplexes by recipient pneumococcal cells (DNA strand resolution-DNA repair-bacterial transformation-genetic recombination).

Heteroduplex molecules of pneumococcal DNA, prepared by cross-annealing resolved complementary strands, have been used as donors in transformation. A number of pairs of genetic markers are situated exclusively in the trans configuration in these heteroduplexes. Insertion of two markers from trans configuration into opposite DNA strands of a recipient genome should result in their segregation after one replication cycle. As a consequence, doubly transformed progeny would not appear, or would be markedly decreased. Contrary to these expectations, transformations with the heteroduplex DNAs give as many doubly transformed progeny for unlinked marker pairs as do homoduplex DNAs. For a pair of markers that normally are weakly linked, the frequencies of cotransfer are actually greater than those observed for a mixture of two singly marked homoduplex DNAs. These results lead to the conclusion that the heteroduplex DNAs are acted upon by repair enzymes of recipient cells so that markers introduced in the trans configuration are frequently converted to the cis configuration, either before or during integration. The efficiency of this conversion suggests that the repair and integration processes may be intimately connected. It is also concluded that complete breakdown of one strand of donor heteroduplex DNA does not occur during DNA uptake by recipient cells.

Centrifugation, Density Gradient↗
PubMed1974

Transformation by simian virus 40 of spleen cells from a hyperimmune rabbit: evidence for synthesis of immunoglobulin by the transformed cells.

Spleen cells derived from a rabbit hyperimmunized with a Type III pneumococcal vaccine were exposed to simian virus 40 in vitro. After 114 days, transformed cells growing on the surface of one culture dish were observed and a cell line was established. The transformed cells had a morphology characteristic of cells transformed by simian virus 40, contained the simian virus 40-specific T antigen, and yielded infectious simian virus 40 upon cultivation with indicator cells in the presence of Sendai fusion factor. Transformed cells incorporated labeled amino acid into protein with the antigenic properties of rabbit immunoglobulin G.

Animals↗
PubMed1977

Cellular transformation and differentiation. Effect of Rous sarcoma virus transformation on sulfated proteoglycan synthesis by chicken chondrocytes.

Incorporation of sulfate into sulfated proteoglycans by isolated chicken chondrocytes was inhibited up to 74% by transformation with the Rous sarcoma virus, and a similar inhibitory effect was observed on acetate incorporation into chondroitin sulfate. Slower sedimenting sulfated proteoglycans appear after the viral transformation. The ratio of chondroitin 4-sulfate to chondroitin 6-sulfate in these slower sedimenting sulfated proteoglycans was different from that of normal chondrocytes, but the chain lengths of sulfated glycosaminoglycans produced by normal chondrocytes and transformed chondrocytes were not significantly different. Chondrocytes were also infected with a temperature-sensitive mutant of RSV, ts LA24, which has a temperature-sensitive lesion in the transforming gene. Hyaluronic acid production by these cells was increased, and the slower sedimenting sulfated proteoglycan was produced only at the permissive temperature.

Acetates↗