Isolation and characterization of plant genomic DNA sequences via (inverse) PCR amplification.
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Base Sequence↗
Biomedical subjects
R Offringa
Publications and source records attributed to R Offringa.
Non-recombinant background in gene targeting: illegitimate recombination between a hpt gene and a defective 5' deleted nptII gene can restore a Kmr phenotype in tobacco.
Previously we have demonstrated gene targeting in plants after Agrobacterium-mediated transformation. In these initial experiments a transgenic tobacco line 104 containing a T-DNA insertion with a defective neomycin phosphotransferase (nptII) gene was transformed with a repair construct containing an otherwise defective nptII gene. Homologous recombination between the chromosomally located target and the incoming complementary defective nptII construct generated an intact nptII gene and led to a kanamycin-resistant (Kmr) phenotype. The gene targeting frequency was 1 x 10(-5). In order to compare direct gene transfer and Agrobacterium-mediated transformation with respect to gene targeting we transformed the same transgenic tobacco line 104 via electroporation. A total of 1.35 x 10(8) protoplasts were transformed with the repair construct. Out of nearly 221,000 transformed cells 477 Kmr calli were selected. Screening the Kmr calli via PCR for recombination events revealed that in none of these calli gene targeting had occurred. To establish the origin of the high number of Kmr calli in which gene targeting had not occurred we analysed plants regenerated from 24 Kmr calli via PCR and sequence analysis. This revealed that in 21 out of 24 plants analysed the 5'-deleted nptII gene was fused to the hygromycin phosphotransferase (hpt) gene that was also present on the repair construct. Sequence analysis of 7 hpt/nptII gene fusions showed that they all contained a continuous open reading frame. The absence of significant homology at the fusion site indicated that fusion occurred via a process of illegitimate recombination. Therefore, illegitimate recombination between an introduced defective gene and another gene present on the repair construct or the chromosome has to be taken into account as a standard byproduct in gene targeting experiments.
Amino Acid Sequence↗
Tumor rejection antigens and tumor specific cytotoxic T lymphocytes.
In several tumor models and in certain types of human malignancies, T cell mediated immune responses can be involved in the host's defences against cancer. Adoptively transferred tumor-specific T cells can mediate complete tumor regression in several animal models and the first effective therapeutic interventions with adoptively transferred (virus-specific) CTL in man have been reported. With the identification of tumor rejection antigens new antigen specific therapeutic approaches come into sight. The development of anti-tumor vaccines based upon known tumor rejection antigens might take off rapidly in the near future, since only the first few tumor rejection antigens of an enormous potential have been reported. This development will go hand in hand with the utilization of cytokines in these vaccines. Cytokines might promote T cell activity or antigen presentation, but can also be used to counteract tumor immune escape mechanisms. Escape from immune surveillance and T cell mediated elimination by tumor cells might account for the failure of the immune system to eradicate or prevent the outgrowth of many types of tumors. The identification of tumor rejection antigens and tumor-specific immune responses, together with a better understanding of the mechanisms by which tumors escape from T cell mediated immunity brings fresh encouragement in the field of tumor immunology.
Animals↗
Nonreciprocal homologous recombination between Agrobacterium transferred DNA and a plant chromosomal locus.
Previously, we demonstrated the occurrence of gene targeting in tobacco cells after Agrobacterium-mediated transformation. In these experiments a defective kanamycin resistance (Kmr) gene residing at a chromosomal location was restored via homologous recombination with an incoming transferred DNA (T-DNA) repair construct (pSDM101) containing a different defective Kmr gene. In this article we describe gene targeting experiments with the same target line, but using an improved repair construct, pSDM321. In one of the Kmr calli obtained after transformation with pSDM321 (line A) the product of homologous recombination was detected using PCR. Further molecular analysis revealed that the defective Kmr gene present on the incoming T-DNA had been restored via homologous recombination with the target locus. The target locus was left unchanged and the corrected T-DNA was found to be inserted on the same chromosome but not close to the target locus. This paper presents molecular evidence in plants for the conversion of an introduced DNA molecule (in this case, T-DNA) by a homologous chromosomal locus.
Base Sequence↗
The thyroid hormone receptor interferes with transcriptional activation via the AP-1 complex.
The nuclear thyroid hormone (T3) receptor, encoded by the c-erbA genes, represses transcriptional activation by the transcription factor AP-1 in a T3-dependent fashion. The viral homologue of the T3 receptor, the v-erbA gene product, does not repress AP-1 activity. Inhibition by T3 involves reduced binding of AP-1 to its cognate DNA target sequence. The reduction in AP-1 binding does not, however, result from competitive binding by the T3 receptor to the AP-1 response element.
Base Sequence↗
Association of CD2 with tubulin. Evidence for a role of the cytoskeleton in T cell activation.
In addition to the antigen-specific T cell receptor, a number of other T cell surface molecules contribute to T cell adhesion and activation. CD2 is a 50-kDa glycoprotein found on the surface of human T lymphocytes which plays a role in T cell adhesion and stimulation. The intracellular mechanisms by which CD2 functions are, however, not fully defined. Here we show that the T cell surface molecule CD2 physically interacts with tubulin. This interaction appears to involve the membrane-proximal part of the cytoplasmic domain of the CD2 molecule, suggesting that CD2 binds to the tubulin fraction previously shown to be present in membranes. Stimulation of T cells with activating pairs of anti-CD2 antibodies, capable of initiating lymphokine production and T cell proliferation, disrupts the CD2-tubulin complexes, suggesting that the dynamic interaction of CD2 with tubulin may play a role in T cell activation.
Amino Acids↗
Adenovirus E1A negatively and positively modulates transcription of AP-1 dependent genes by dimer-specific regulation of the DNA binding and transactivation activities of Jun.
Adenovirus E1A proteins inhibit expression of the collagenase gene but activate expression of the c-jun gene. Both effects are mediated by TPA-responsive elements (TREs), the binding sites for members of the AP-1 transcription factor family. By a process that is independent of the retinoblastoma gene product, E1A distinguishes between different AP-1 factors: in vivo binding of Jun/Jun homodimers and Jun/Fos heterodimers to the collagenase TRE is totally blocked by E1A while, in contrast, there is no inhibition of Jun/ATF-2 binding to the TRE sequences in the c-jun promoter. Altered phosphorylation of the DNA binding domain of cJun is not involved in the inhibition of cJun/cJun and cJun/cFos binding. E1A does, however, cause hyperphosphorylation of the transactivation domain of cJun, which is likely to be responsible for the enhanced c-jun transcription by E1A mediated through cJun/ATF-2 heterodimers.
Activating Transcription Factors↗
Mechanisms of intermolecular homologous recombination in plants as studied with single- and double-stranded DNA molecules.
To elucidate the mechanism for intermolecular homologous recombination in plants we cotransformed Nicotiana tabacum cv Petit Havana SR1 protoplasts with constructs carrying different defective derivatives of the NPTII gene. The resulting kanamycin resistant clones were screened for possible recombination products by PCR, which proved to be a valuable technique for this analysis. Our results show that the double-stranded circular DNA molecules used in this study recombine predominantly via a pathway consistent with the single-strand annealing (SSA) model as proposed for extrachromosomal recombination in mammalian cells. In the remaining cases recombination occurred via a single reciprocal recombination, gene conversion and possibly double reciprocal recombination. Since single-stranded DNA is considered to be an important intermediate in homologous recombination we also established the recombination ability of single-stranded DNA in intermolecular recombination. We found that single-stranded DNA enters in recombination processes more efficiently than the corresponding double-stranded DNA. This was also reflected in the recombination mechanisms that generated the functional NPTII gene. Recombination between a single-stranded DNA and the complementing DNA duplex occurred at similar rates via a single reciprocal recombination and the SSA pathway.
Base Sequence↗
Regulation of parathyroid hormonelike protein production in cultured normal and malignant keratinocytes.
We have recently demonstrated that parathyroid hormone-like protein (PLP) production by cultured human squamous carcinoma cells (SCC) can be modulated by co-culture with fibroblasts. The interaction of SCC with fibroblasts, possibly occurring during the invasive phase of SCC, may be the stimulus for enhanced PLP production, thus contributing to the genesis of humoral hypercalcemia of malignancy in this type of cancer (Cancer Res 50:3589-3594, 1990). In the present study we show that the fibroblast-induced increase in PLP level in the medium of SCC-4 cells is paralleled by an increase in PLP messenger ribonucleic acid (mRNA) expression in these cells. We also found that the inhibition of secretion of PLP by monensin for 2 h resulted in a marked increase in immunodetectable PLP intracellularly, suggesting that secretion of PLP was a fast process. The modulation of the production of PLP by calcium and hydrocortisone was further examined in SCC-4 cells and was compared to that in normal keratinocytes and in SCC-9 cells. PLP levels in conditioned media were highest in poorly differentiating SCC-4 cells, intermediate in moderately differentiating SCC-9 cells, and lowest in normal keratinocytes showing high differentiating capacity. Furthermore, in each of the cell types used, PLP production was highest in cultures grown under low calcium conditions; at both calcium concentrations used, the presence of hydrocortisone reduced the PLP release into the medium. This reduction was probably due to a direct effect of hydrocortisone on PLP synthesis because the expression of PLP mRNA was also reduced in the presence of hydrocortisone when tested in SCC-4 cells. In conclusion, our findings indicate that the induction of differentiation in both normal and malignant keratinocytes is associated with the inhibition of PLP production.
Carcinoma, Squamous Cell↗
A quick method to estimate the T-DNA copy number in transgenic plants at an early stage after transformation, using inverse PCR.
Agrobacterium-mediated transformation of plants is known to result in transgenic plants with a variable number of integrated T-DNA copies. Our aim was to obtain transgenic tobacco plants containing one integrated T-DNA copy per genome. Therefore, a quick method was developed to estimate the T-DNA copy number of young transgenic plantlets within 10 weeks after transformation. Inverse polymerase chain reaction (IPCR) was used to amplify junction fragments, i.e. plant genomic DNA sequences flanking the known T-DNA sequences.
Base Sequence↗
A novel function of the transforming domain of E1a: repression of AP-1 activity.
Adenovirus E1a represses transcription of the collagenase gene via the phorbol ester-responsive element (collTRE). The mechanism involves inhibition of the trans-activating function of the transcription factor AP-1 without reduction of its synthesis and without any apparent change in DNA binding or composition. The ability of E1a to downmodulate AP-1 is a unique property among dominant oncogenes. This repression depends on conserved region 1, one of the transforming domains of E1a, indicating that it is an integral feature of adenovirus transformation.
Adenovirus Early Proteins↗
Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation.
We determined whether T-DNA molecules introduced into plant cells using Agrobacterium are suitable substrates for homologous recombination. For the detection of such recombination events different mutant versions of a NPTII construct were used. In a first set of experiments protoplasts of Nicotiana tabacum SR1 were cocultivated with two Agrobacterium tumefaciens strains. Each strain contained a different T-DNA, one carrying a 5' deleted NPTII gene and the other a NPTII gene with a 3' deletion. A restored NPTII gene was found in 1-4% of the protoplasts that had been cotransformed with both T-DNAs. Restoration of the NPTII gene could only be the consequence of homologous recombination between the two different T-DNAs in the plant cell, since the possibility of recombination in Agrobacterium was excluded in control experiments. In subsequent experiments was investigated the potential use of Agrobacterium for gene targeting in plants. A transgenic tobacco line with a T-DNA insertion carrying a defective NPTII gene with a 3' deletion was transformed via Agrobacterium with a T-DNA containing a defective NPTII repair gene. Several kanamycin resistant plant lines were obtained with an intact NPTII gene integrated in their genome. In one of these lines the defective NPTII gene at the target locus had been properly restored. Our results show that in plants recombination can occur between a chromosomal locus and a homologous T-DNA introduced via A. tumefaciens. This opens the possibility of using the Agrobacterium transformation system for site directed mutagenesis of the plant genome.
Blotting, Southern↗
Differential effects of the adenovirus E1A oncogene on members of the AP-1 transcription factor family.
The adenovirus early region 1A (E1A) oncogene interferes with the expression level and activity of the AP-1 transcription factor family. E1A abolished the transactivating function of AP-1 (Jun/Fos), which binds to the 12-O-tetradecanoylphorbol-13-acetate-responsive element of the collagenase gene (collTRE). In contrast, the activity of another member of the AP-1 family that binds to the c-junTRE was not repressed. The mRNA expression of the c-jun gene was, in fact, strongly elevated in various cell types expressing the E1A gene of either adenovirus type 5 (Ad5) or Ad12. The regulation of the junB gene by adenovirus E1A, on the other hand, depended both on the cell type and on the transforming adenovirus serotype. The fact that E1A-induced alterations in the repertoire of AP-1 transcription factors depend on its transforming domain in conserved region 1 suggests that the effects are relevant for the transformation process.
Adenovirus Early Proteins↗
Eradication of adenovirus E1-induced tumors by E1A-specific cytotoxic T lymphocytes.
Cytotoxic T lymphocyte (CTL) clones against adenovirus type 5 (Ad5) early region 1 (E1)-transformed cells were generated in C57BL/6 (B6) mice. A defined peptide encoded by Ad5 E1A is the target structure for H-2Db-restricted CTLs. Upon intravenous injection into B6 nude mice bearing Ad5 E1-induced tumors, these CTLs, if combined with recombinant IL-2, destroy subcutaneous tumor masses up to 10 cm3. The in vivo action of CTLs is highly specific, and long-term "memory" persists in treated nude mice months after tumor regression. Our data show an important role for CTLs directed against a viral nuclear oncogene product in tumor eradication.
Adenoviridae Infections↗
Suppression of cellular gene activity in adenovirus-transformed cells.
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Adenoviridae↗
Immunosurveillance of virus-induced tumors.
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Adenoviruses, Human↗
The repression of the growth factor-inducible genes JE, c-myc and stromelysin by adenovirus E1A is mediated by conserved region 1.
The growth factor-inducible cellular genes JE, c-myc and stromelysin (sml) are strongly repressed upon transformation by adenovirus E1A. As E1A proteins are multifunctional and apparently contain distinct domains (conserved regions 1, 2 and 3), each with a specific effect on gene regulation and cell-transformation, we have investigated which of the three conserved regions are responsible for the reduced expression of these genes. To this end, we monitored the expression of the JE, sml and c-myc genes in a panel of normal rat kidney (NRK) cells expressing different mutant E1A genes. Only CR1, and not CR2 or CR3 were found to be essential for the repression of the genes, indicating that CR1, one of the regions essential for cell transformation, represents an autonomous gene regulatory function that can operate in the absence of CR2. We also show that the association of E1A proteins to a 300 kD cellular protein in NRK cells coincides with the ability to repress these genes.
Adenovirus Early Proteins↗
Similar effects of adenovirus E1A and glucocorticoid hormones on the expression of the metalloprotease stromelysin.
The stromelysin (sml) gene encodes a secreted protease which degrades components of the extracellular matrix. Transformation of NRK49F cells by the E1A region of adenovirus (Ad) type 5 or 12 reduces sml RNA levels, whereas various growth factors or EJras-mediated transformation stimulate sml gene expression in these cells. Nuclear run-on experiments show that AdE1A, growth factors and EJras act on sml gene expression at the level of transcription. Although the sml gene is strongly suppressed in AdE1A-transformed cells, treatment with growth factors or transfection of EJras still causes a raise in sml mRNA levels, indicating that E1A does not block the induction mechanism itself. The effect of glucocorticoid hormones on sml gene expression is very similar to that of AdE1A, in that mRNA levels are lowered without affecting the induction phenomenon. This similarity may provide a clue to the mechanism by which AdE1A represses cellular gene activity.
Adenoviruses, Human↗