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PubMed1999

Transformation of Candida oleophila and survival of a transformant on orange fruit under field conditions.

Histidine auxotrophs of wild-type strain I-182 of Candida oleophila, produced using ethyl methanesulfonate, were transformed with plasmids containing the HIS3, HIS4 and HIS5 genes of Saccharomyces cerevisiae. Histidine auxotrophy was complemented by the HIS5 gene of S. cerevisiae. Stability of the transformants under non-selective conditions and DNA gel-blot analysis suggested that the transforming DNA had integrated into the C. oleophila genome. There were no detectable physiological differences between the wild-type and the transformants. The biological control ability of C. oleophila was not affected by the transformation. A genetically marked transformant (with a beta-glucuronidase gene) colonized wounds on oranges, and its population increased under field conditions. The identity of the genetically marked transformant was established by PCR-amplification of a portion of the beta-glucuronidase gene.

2-Aminoadipic Acid↗
PubMed2003

A new transformation-regeneration procedure in the model legume Lotus japonicus: root explants as a source of large numbers of cells susceptible to Agrobacterium-mediated transformation.

We describe herein a simple and efficient transformation procedure for the production of transgenic Lotus japonicus plants. In this new procedure, dedifferentiated root explants, used as starting material, are the source of a large number of cells that are competent for the regeneration procedure, with a high susceptibility to Agrobacterium infection. The application of this protocol resulted in a tenfold increase in the number of transformants produced by a single plant in comparison to the widely used hypocotyl transformation procedure. Furthermore, our procedure allowed the use of intact plants stored for a long time at 4 degrees C, thus providing a potential continuous supply of explants for transformation experiments. The overall time of incubation under tissue culture conditions required to obtain a plant transferable into soil is 4 months. The transgenic nature of the transformants was demonstrated by the detection of beta-glucuronidase (GUS) activity in the primary transformants and by molecular analysis. Stable transformation was indicated by Mendelian segregation of the hygromycin selectable marker and of the gusA activity after selfing of the transgenic plants.

Agrobacterium tumefaciens↗
PubMed2005

Stable transformation and direct regeneration in Coffea canephora P ex. Fr. by Agrobacterium rhizogenes mediated transformation without hairy-root phenotype.

A system for genetic transformation of Coffea canephora by co-cultivation with Agrobacterium rhizogenes harbouring a binary vector has been developed. The objective of the present study was the genetic transformation and direct regeneration of transformants through secondary embryos bypassing an intervening hairy root stage. Transformants were obtained with a transformation efficiency up to 3% depending on the medium adjuvant used. A. rhizogenes strain A4 harbouring plasmid pCAMBIA 1301 with an intron uidA reporter and hygromycin phosphotransferase (hptII) marker gene was used for sonication-assisted transformation of Coffea canephora. The use of hygromycin in the secondary embryo induction medium allowed the selection of transgenic secondary embryos having Ri T-DNA along with the T-DNA from the pCAMBIA 1301 binary vector. In addition transgenic secondary embryos devoid of Ri-T-DNA but with stable integration of the T-DNA from the binary vector were obtained. The putative transformants were positive for the expression of the uidA gene. PCR and Southern blot analysis confirmed the independent, transgenic nature of the analysed plants and indicated single and multiple locus integrations. The study clearly demonstrates that A. rhizogenes can be used for delivering transgenes into tree species like Coffea using binary vectors with Agrobacterium tumefaciens T-DNA borders.

Blotting, Southern↗
PubMed2004

Transformation of Fusarium oxysporum by particle bombardment and characterisation of the resulting transformants expressing a GFP transgene.

Fusarium is the causative agent of a variety of economically significant vascular wilt diseases of vegetables, flowers and field crops. The completion of the first Fusarium genome and the availability of an EST database now provides a platform for both forward and reverse genetic approaches to ascribe gene function in this phytopathogen. To underpin these strategies effective gene transfer procedures will be required. Here we describe an efficient and robust procedure for Fusarium oxysporum transformation based on particle bombardment. We utilised this procedure to introduce a chimeric gene comprised of the Aspergillus nidulans Pgdp promoter fused to a GFP reporter gene. A transformation efficiency of 45 transformants per mug of plasmid DNA was routinely achieved. The Pgdp promoter directed strong cytoplasmic expression of the GFP marker in transformed F. oxysporum monitored via fluorescence and confocal microscopy. A pathogenicity assay undertaken on Arabidopsis seedlings with selected transformants revealed that virulence was retained following transformation. Moreover, in a similar fashion to wild-type F. oxysporum, these transformants activated three distinct Arabidopsis defence gene promoter::luciferase fusions, which defined specific defence gene subsets.

Arabidopsis↗
PubMed1993

Energy metabolism of non-transformed and benzypyrene-transformed 3T3 cells: a microcalorimetric study.

Non-transformed and benzpyrene-transformed 3T3 cells attached to microcarriers were studied with microcalorimetry. The transformed cells manifested a higher rate of heat production and a larger anaerobic contribution to the total catabolism than did the non-transformed cells. Moreover, the transformed cells were characterized by a highly pH-dependent metabolism with a drop in thermal power occurring 0.3 pH units lower than the corresponding drop for the non-transformed cell line. We believe this shift in pH to be due to the higher intracellular pH maintained by the transformed cells. In addition, the study describes the correlation between rate of heat production per cell and the degree of confluency for each cell line, respectively.

3T3 Cells↗
PubMed1986

Intracellular K+ and the expression of transformation parameters by chick cells transformed with the Bryan strain of Rous sarcoma virus.

As normal chick embryo (CE) cells entered quiescence the intracellular concentrations of both Na+ and K+ declined. Comparable decreases in intracellular concentrations of Na+ and K+ were not observed in CE cells transformed by either the Schmidt-Ruppin (SR) or the Bryan (B) strain of Rous sarcoma virus (RSV). Intracellular concentrations of Na+ were higher in SR-RSV-transformed CE cells than in B-RSV-transformed cells and uninfected CE cells at all times after plating. In contrast, intracellular concentrations of K+ were higher in B-RSV-transformed CE cells than in SR-RSV-transformed cells. Uninfected CE cells incubated in medium containing an elevated concentration of K+ (an increase from 5 to 30 mM) exhibited several, but not all, of the transformation parameters expressed by B-RSV-transformed CE cells.

Animals↗
PubMed1988

"Hit and run" transformation of mouse C127 cells by bovine papillomavirus type 4: the viral DNA is required for the initiation but not for maintenance of the transformed phenotype.

Morphological transformation of C127 mouse fibroblasts by bovine papillomavirus type 4 (BPV-4) DNA depends on additional factors, including cell density, the presence of TPA, the concentration of fetal calf serum, and the physical state of the input DNA. Low cell density or the presence of TPA allows the achievement of full transformation, suggesting that disturbance of cell-to-cell contact may be necessary for the expression of the malignant phenotype. TPA also induces a burst of viral DNA synthesis and transcription. The frequency of focus formation is fivefold higher in the presence of high serum than in low serum, indicating that growth factors are required during the early stages of transformation. Although the same number of foci is obtained with either circular or linear DNA, the cells transfected with linear DNA are at a more advanced stage of transformation, being capable of anchorage-independent growth and tumorigenic in nude mice; only 5 out of 21 of these cell lines contain viral DNA in integrated and rearranged forms. In contrast cells transfected with circular DNA are anchorage-dependent but tumorigenic in mice, and do not contain any viral DNA. The linear DNA has an interrupted E1 ORF and fully transformed cells are obtained also with a subgenomic viral DNA fragment containing part of the L1 ORF, the complete E6, E7, and E8 ORFs, and the truncated E1 ORF under the control of the BPV-4 regulatory region; viral DNA is not maintained in any of these cell lines. In total, only 15% of the transformed cell lines contains viral DNA. These results are discussed in the light of the "hit and run" model of cell transformation.

Animals↗
PubMed1979

Tumor antigens of human Ad5 in transformed cells and in cells infected with transformation-defective host-range mutants.

We have studied the polypeptides associated with the expression of the transforming region of the Ad5 genome by immunoprecipitating antigens (using the double antibody and protein A-Sepharose techniques) from cells infected with wild-type (wt) Ad5 or transformation-defective host range (hr) mutants and from cells transformed by Ad5. Three different antisera were used: P antiserum specific for early viral products (Russell et al., 1967) and two different hamster tumor antisera. Immunoprecipitation of antigens from wt-infected KB cells followed by SDS-polyacrylamide gel electrophoresis of precipitated proteins revealed that a major polypeptide having a molecular weight of approximately 58,000 was detected with all three antisera and with both the double antibody and the protein A-Sepharose techniques, while P antiserum also precipitated polypeptides of molecular weights 72,000, 67,000 and 44,000, which probably represent the DNA binding protein and related polypeptides, respectively. With the double antibody technique, in addition to the proteins mentioned above, P antiserum and the hamster tumor antisera precipitated a 10,500 dalton polypeptide which was not detected when the protein A-Sepharose procedure was used. Using either the double antibody or the protein A-Sepharose technique, we found that hr mutants from complementation group II failed to induce the synthesis of the 58,000 dalton protein, whereas mutants from complementation group I produced normal or near normal amounts. Using the double antibody technique, we found that the 10,500 dalton protein was absent or made in reduced amounts by group I mutants. A 58,000 dalton protein was detected in a number of different Ad5-transformed cell lines, including the 293 human line, the 14b hamster line and several transformed rat cell lines. This observation and the fact that transformation negative group II mutants fail to induce the synthesis of a 58,000 dalton polypeptide suggest that this protein is one of the Ad5-specific products necessary for cell transformation.

Adenoviruses, Human↗
PubMed2005

Caffeic acid phenethyl ester (CAPE) prevents transformation of human cells by arsenite (As) and suppresses growth of As-transformed cells.

Recent evidence suggests that inflammatory cytokines and growth factors contribute to arsenite (As)-induced human carcinogenesis. We investigated the expression of inflammatory cytokine mRNAs during the transformation process induced by chronic As exposure in non-tumorigenic human osteogenic sarcoma (N-HOS) cells using gene arrays, and results were confirmed by RT-PCR and protein arrays. Caffeic acid phenethyl ester (CAPE), a naturally occurring immunomodulating agent, was used to evaluate the role of inflammatory factors in the process of As-mediated N-HOS cell transformation and in As-transformed HOS (AsT-HOS) cells. We found that an 8-week continuous exposure of N-HOS to 0.3 microM arsenite resulted in HOS cell transformation. That exposure also caused substantial decreases in inflammatory cytokine mRNAs, such as interleukin (IL) IL-1alpha, IL-2, IL-8, IL-18, MCP-1, TGF-beta2, and TNF-alpha, while it increased c-jun mRNA in a time-dependent manner. Co-incubation of N-HOS with As and CAPE (0.5-2.5 microM) prevented As-mediated declines in cytokine mRNAs in the co-treated cells, as well as their transformation to anchorage independence, while it caused decreases in c-jun mRNA. CAPE (up to 10 microM) had no effect on growth of N-HOS cells. However, CAPE (1-10 microM) treatment of AsT-HOS cells inhibited cell growth, induced cell cycle G2/M arrest, and triggered apoptosis, accompanied by changes in cytokine gene expression, as well as decreases in cyclin B1 and cdc2 abundance. Resveratrol (RV) and (-)(.) epigallocatechin gallate (EGCG), preventive agents present in grapes and green tea, respectively, induced similar changes in AsT-HOS cell growth but required much higher doses than CAPE to cause 50% growth arrest (<2.5 microM CAPE versus 25 microM RV or 50 microM EGCG). Overall, our findings suggest that inflammatory cytokines play an important role in the suppressive effects of CAPE on As-induced cell transformation and in the selective cytotoxicity of CAPE to As-transformed HOS cells.

Antioxidants↗
PubMed1997

The S3a ribosomal protein gene is identical to the Fte-1 (v-fos transformation effector) gene and the TNF-alpha-induced TU-11 gene, and its transcript level is altered in transformed and tumor cells.

Previous work on mouse x rat hybrid cells (BS series) led to the assignment of a transformation suppressor locus (Sail) to the rat 5q22-q33 region. This gene is not yet identified. From a non-transformed BS hybrid cell line, we isolated a partial cDNA insert (13T), which detects a transcript more abundant in transformed cells than in their non-transformed homologs. Sequence comparisons led us to conclude that 13T is identical to the coding sequences of the ribosomal protein S3a gene (Rps3a), of Fte-1 (v-fos transformation effector gene) and of TU-11, a mouse gene induced by TNF-alpha. Rps3a, Fte-1 and TU-11 are thus one and the same gene. Similarity was also found between this gene and non-mammalian sequences reported to be involved in cell cycling. Like the Rps3a transcript level, the c-Fos transcript level is higher in transformed cells. Rps3a and Fos could thus be effectors of the transformed phenotype.

Animals↗
PubMed1991

Epithelial-mesenchymal transformation of embryonic cardiac endothelial cells is inhibited by a modified antisense oligodeoxynucleotide to transforming growth factor beta 3.

During early cardiac development, the progenitor cells of the heart valves and membranous septa undergo an epithelial-mesenchymal transformation. Previous studies have shown that this transformation depends on the activity of a transforming growth factor beta (TGF beta) molecule produced by the heart. In the present study, we have used modified antisense oligodeoxynucleotides generated to nonconserved regions of TGF beta 1, -2, -3, and -4 to examine the possible roles of these members in this transformation. A phosphoramidate-modified oligonucleotide complementary to TGF beta 3 mRNA was capable of inhibiting normal epithelial-mesenchymal transformation by 80%. Unmodified oligonucleotides to TGF beta 3, modified oligonucleotides to TGF beta 1, -2, and -4, and two modified control oligonucleotides were unable to inhibit the transformation. These data demonstrate that a specific member of the TGF beta family, TGF beta 3, is essential for the epithelial-mesenchymal cell transformation.

Animals↗
PubMed1992

A mutant p53 protein is required for maintenance of the transformed phenotype in cells transformed with p53 plus ras cDNAs.

Mutant p53 and activated ras cDNA clones cooperate to fully transform primary rat embryo fibroblasts in cell culture, whereas neither cDNA alone results in the full transformation of these cells. The mutant p53 protein may be required to initiate the transformation event with ras. Alternatively, mutant p53 gene expression may be required to maintain the properties of the transformed phenotype. To distinguish between these possibilities, primary rat embryo fibroblasts were transformed with mutant p53 plus ras cDNAs, where the expression of the p53 gene was regulated by an isopropyl beta-D-thiogalactoside-responsive promoter. When expression of the mutant p53 cDNA was inhibited and no detectable exogenous p53 protein was produced, both the growth rate and the morphology of the cells reverted to a normal phenotype. These results demonstrate that a mutant p53 protein is required for the maintenance of the transformed phenotype in cells transformed with p53 plus ras cDNAs.

Animals↗
PubMed1994

Cellular epigenetics: control of the size, shape, and spatial distribution of transformed foci by interactions between the transformed and nontransformed cells.

NIH 3T3 cells that are passaged frequently at low density in high (10%) calf serum lose their original capacity to produce transformed foci on a monolayer of nontransformed cells. They can then be used to form a monolayered background for the assay of the number of focus-forming cells from a transformed population. Continuation of the low-density passages for many weeks gives rise to a population that can suppress the full development of foci by a transformed line. The suppression appears to occur only after the background cells have become confluent and contact inhibited. It can also cause the disappearance of light foci that had developed before suppression began. Another subline of cells that were passaged at cloning density only once a week lose their focus-forming capacity more slowly than those passaged thrice weekly. When used as a background for the assay of a transformed line, they permit continuous expansion of the foci, with no sign of suppression. Not only the number and size of foci but also their detailed morphology is influenced by the background on which they are formed. A suppressive background can also determine the spatial distribution of foci, presumably as a result of gradients in local cell density of the background. The permissiveness of a nontransformed cell population for focus formation by transformed cells appears to be related to the capacity of the nontransformed population itself to undergo transformation when exposed to the constraints used to induce transformation. These findings indicate there are many degrees of capacity to suppress focus formation and to overcome suppression. They have significance for tumor development and for the epigenetic interactions of normal development.

3T3 Cells↗
PubMed2000

Pituitary tumor transforming gene (PTTG) transforming and transactivation activity.

Pituitary tumor transforming gene (PTTG) is a newly identified transforming gene, the functional mechanism of which is little understood. Computational analysis reveals a C terminus rich in Glu and Pro, a known characteristic of transcriptional activation domains. We report here that murine PTTG indeed possesses transactivation ability, which correlates highly with its transforming properties. Pro(139), Ser(159), Pro(157)-Pro(158)-Ser(159)-Pro(160) (PPXP motif), and Leu(120)-Asp(121)-Phe(122)-Asp(123)-Leu(124) were found to be important for transactivation. Mutation to Ala at a key Pro(139) residue not only disrupted the transactivation function but also resulted in the loss of transforming ability in NIH3T3 cells. A murine PTTG cDNA that encodes a variant C-terminal tail (Gly-Lys-Gly-Val-Arg-Ser-Asn-Gly-Cys-Lys-Asp-Leu-Val-Thr) was cloned. This novel PTTG is devoid of transactivation and transforming ability; deletion of its variant C-terminal tail restores both transactivation and transforming ability. These results show a high correlation between the transforming and transactivation functions of PTTG and also indicate that the novel PTTG variant may function as an endogenous competitor to wild-type PTTG.

Amino Acid Sequence↗
PubMed2001

Hepatitis B virus X protein differentially activates RAS-RAF-MAPK and JNK pathways in X-transforming versus non-transforming AML12 hepatocytes.

The hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis of chronic HBV patients by an unknown mechanism. Activities of pX likely relevant to hepatocyte transformation include activation of the mitogenic RAS-RAF-MAPK and JNK pathways. To assess the importance of mitogenic pathway activation by pX in transformation, we employed a cellular model system composed of two tetracycline-regulated, pX-expressing cell lines, constructed in AML12-immortalized hepatocytes. This system includes the differentiated 3pX-1 and the de-differentiated 4pX-1 hepatocytes. Our studies have demonstrated that conditional pX expression transforms only 3pX-1 cells. Here, comparative in vitro kinase assays and various in vivo analyses demonstrate that pX affects an inverse activation of RAS-RAF-MAPK and JNK pathways in 3pX-1 versus 4pX-1 cells. Sustained pX-dependent RAS-RAF-MAPK pathway activation is observed in pX-transforming 3pX-1 cells, whereas sustained pX-dependent JNK pathway activation is observed in pX non-transforming 4pX-1 cells. This differential, pX-dependent mitogenic pathway activation affects differential activation of cAMP-response element-binding protein and c-Jun and determines the proliferative response of 3pX-1 and 4pX-1 cells. Furthermore, tetracycline-regulated, pX-NLS-expressing cell lines demonstrate that expression of the nuclear pX-NLS variant minimally activates the RAS-RAF-MAPK pathway and results in markedly reduced transformation. These results link sustained, pX-mediated activation of RAS-RAF-MAPK pathway to hepatocyte transformation.

Cell Division↗
PubMed2002

Transformation of Bacillus subtilis in chocolate milk: evidence for low frequency of establishment of cells transformed under non-selective conditions.

Transformation of naturally competent Bacillus subtilis with plasmid was carried out in chocolate milk without antibiotics. Transformed cells were enumerated during the entire growth phase in chocolate milk. When DNA was added to aliquots of a batch culture after different times of incubation, transformation events were detected at all different growth stages. When DNA was added to a batch culture together with the inoculum, transformed cells were detected at the onset of exponential growth. However, apparently no or only limited growth of these transformed cells was observed. To clarify, whether the limitation of growth was due to suppression by non-transformed cells, different proportions of B. subtilis cells either carrying or not carrying the plasmid were mixed and inoculated into chocolate milk without antibiotic. Our results indicate that suppression appears to be of minor importance. Instead, plasmid-bearing cells appear to suffer from a prolonged lag-phase. However, the failure to exhibit significant growth of cells which had taken up the plasmid in chocolate milk appears to be due to failure of these cells to establish themselves as permanently transformed under non-selective conditions.

Animals↗
PubMed1977

Cyclic AMP-induced morphological transformation of cells infected by temperature-sensitive mouse sarcoma virus. Expression of transformation-associated markers.

Normal rat kidney (NRK) cells infected with a temperature-sensitive (ts) mutant of mouse sarcoma virus (NRK [MSV-1b]) express the transformed phenotype when grown under permissive conditions, but acquire the normal phenotype when grown under restrictive conditions. Addition of 3', 5' cyclic adenosine monophosphate (cAMP) to NRK (MSV-1b) cells grown at the restrictive temperature results in morphological transformation. To determine whether other markers associated with the transformed phenotype were coordinately expressed after cAMP exposure, concanavalin A (Con A) agglutinability, hexose transport rate, and incorporation of radioactively labeled fucose into fucolipid III and fucolipid IV (FL III and FL IV ) of the cells were examined. NRK cells transformed by wild-type MSV or NRK(MSV- 1b) grown under permissive conditions were agglutinated by low concentrations of Con A and exhibited relatively high maximal agglutination levels which were specifically inhibited by alpha-methyl-D-mannoside. In contrast, NRK (MSV-1b) cells grown under restrictive conditions were weakly agglutinated by Con A and exhibited reduced maximal agglutination levels, similar to uninfected NRK cells. Treatment of NRK (MSV-1b) cells at the restrictive temperature with cAMP resulted in morphological transformation and a change in the pattern of incorporation of labeled fucose inot FL III and FL IV to one comparable to that of NRK (MSV-1b) cells at the permissive temperature or to NRK cells transformed by wild-type MSV. In contrast, cAMP treatment resulted in no increase in Con A agglutinability or 2 deoxy-D- [(3)H]glucose transport relative to mock treated cultures. The results demonstrate that cAMP-induced morphological transformation and altered fucolipid composition of NRK (MSV-1b) cells are not correlated with alterations in hexose transport rate or Con A agglutinability.

Agglutination↗
PubMed1973

An enzymatic function associated with transformation of fibroblasts by oncogenic viruses. II. Mammalian fibroblast cultures transformed by DNA and RNA tumor viruses.

Chick, hamster, mouse, and rat embryo fibroblast cultures, transformed by either DNA or RNA viruses, show fibrinolytic activity under suitable conditions of growth and in appropriate media; normal counterpart cultures do not. The fibrinolysin is produced by the interaction of two protein factors: one of these, a cell factor, is released by transformed cells and accumulates in the medium when cultures are incubated in the absence of scrum. The second factor, the serum factor, is a specific protein that is present in sera of many avian and mammalian species, including man. Not all sera yield fibrinolysin on interaction with any given transformed cell factor, and the spectrum of activating sera is distinctive for each cell factor. This pattern appears to be determined by the cell type, rather than by the transforming virus. An important role for the fibrinolysin in oncogenic transformation is suggested by the following correlations. (a) The initial appearance of fibrinolysin precedes the morphological change after the transfer to permissive temperatures of chick fibroblast cultures infected with a temperature-sensitive mutant of RSV. (b) The initiation of fibrinolysis and of morphological change both require the synthesis of new protein, but not the synthesis of either DNA or rRNA. (c) The activity of the fibrinolysin is correlated with the retention of abnormal morphology in hamster cells transformed by SV-40. (d) The sera of normal chicks effectively activate fibrinolysis with the cell factor from transformed chick cells. In contrast the sera of chicks with RSV tumors do not; these contain an inhibitor of the fibrinolytic activity.

Animals↗