The influence of c-myc expression on fibroblast growth and morphology was investigated by transfection of c-myc genes linked to viral promoters. No foci were observed after transfection of either NIH/3T3 or Rat 2 cells. Cell lines containing activated c-myc genes were established using SV2-neo coselection and several growth parameters of the cells were studied. The cells showed a slight increase in refractility and formed colonies in soft agar with an efficiency of only 1%-2%. The c-myc-transfected cells grew well in 0.5% serum while the controls did not. The major difference in cell growth noted was that c-myc-transfected cells were tumorigenic when inoculated into nude mice or syngeneic rats. Analysis of RNA from the tumorigenic cells showed a level of c-myc expression from the transfected genes that was 2 to 6 fold higher than that from the endogenous gene. The level of c-myc RNA in the fibroblast tumors was similar to that found in mouse plasmacytomas. Expression of the endogenous c-myc gene was unaffected by the transfected genes for subconfluent cells in culture, but the gene was shut off in the nude mouse tumors. These results demonstrate that constitutive c-myc expression leads to tumorigenicity in immortalized cell lines.

The plant genome responds to the bacteriophage P1-derived loxP-Cre site-specific recombination system. Recombination took place at loxP sites stably integrated in the tobacco genome, indicating that the Cre recombinase protein, expressed by a chimeric gene also stably resident in the genome, was able to enter the nucleus and to locate a specific 34 bp DNA sequence. An excisional recombination event was monitored by the acquisition of kanamycin resistance, which resulted from the loss of a polyadenylation signal sequence that interrupted a chimeric neomycin phosphotransferase II gene. Molecular analysis confirmed that the excision had occurred. Recombination occurred when plants with the integrated loxP construction were stably re-transformed with a chimeric cre gene and when plants with the introduced loxP construction were cross-bred with those carrying the chimeric cre gene. As assayed phenotypically, site-specific recombination could be detected in 50%-100% of the plants containing both elements of the system. Kanamycin resistance was detected at 2-3 weeks after re-transformation and in the first leaf of hybrid seedlings. This demonstration of the effectiveness of the loxP-Cre system in plants provides the basis for development of this system for such purposes as directing site-specific integration and regulation of gene expression.

Over 40,000 species of plants accumulate fructan, p-2-1-and P-2-6-linked polymers of fructose as a storage reserve. Due to their high fructose content, severa1 commercial applications for fructans have been proposed. However, plants that accumulate these polymers are not agronomically suited for large-scale cultivation or processing. This study describes the transformation of a Bacillus amyloliguefaciens SacB gene into maize (Zea mays 1.) callus by particle bombardment. Tissue-specific expression and targeting of the SacB protein to endosperm vacuoles resulted in stable accumulation of high-molecular-weight fructan in mature seeds. Accumulation of fructan in the vacuole had no detectable effect on kernel development or germination. Fructan levels were found to be approximately 9-fold higher in sh, mutants compared to wild-type maize kernels. In contrast to vacuole-targeted expression, starch synthesis and endosperm development in mature seeds containing a cytosolically expressed SacB gene were severely affected. The data demonstrate that hexose resulting from cytosolic SacB activity was not utilized for starch synthesis. Transgenic seeds containing a chimeric SacB gene provide further evidence that the dominant pathway for starch synthesis in maize endosperm is through uridine diphosphoglucose catalyzed by the enzyme sucrose synthase.

No abstract

Many recent studies have shown that chromosomal translocation breakpoints frequently occur near cellular proto-oncogenes (reviewed in ref. 1). In both mouse plasmacytomas and Burkitt lymphomas, the c-myc oncogene becomes joined to an immunoglobulin heavy-chain gene in a head-to-head configuration. Within c-myc, the breaks frequently occur near the first exon-intron boundary, while within the immunoglobulin gene the breaks usually involve sequences directing heavy-chain switching. It has been assumed that the translocations represent abortive immunoglobulin switching events which have activated the c-myc gene for a role in tumour formation. However, sequence analysis of the c-myc gene does not reveal any apparent similarity to the immunoglobulin switch signals. With these results in mind, we have determined the precise breakpoints within c-myc for two plasmacytoma lines in order to search for any common features that may shed some light on the mechanism of chromosomal translocation. We report here that the tetranucleotide sequence GAGG occurs close to the breakpoint in five out of six translocations, and so may be a sequence recognized by either the enzymes that catalyse immunoglobulin heavy-chain switching, or some other DNA-cleaving activity.

Genes encoding wild type acetolactate synthase (ALS) and a sulfonylurea herbicide-resistant form of the enzyme, isolated from Arabidopsis thaliana, were expressed in transgenic Nkotiana tabacum plants under the control of their native promoters or of the highly active cauliflower mosaic virus 35S promoter. Expression of the wild type coding region from the 35S promoter resulted in a small, threefold increase in sulfonylurea tolerance above the levels measured in tissue expressing the nafive wild type gene. A much larger, 300-fold increase in herbicide tolerance was conferred by the mutant gene encoding a herbicide-resistant ALS. An additional 10-fold increase in tolerance was attained by expressing this coding region from the 35S promoter. The increase in both wild type and mutant gene expression directed by the 35S promoter resulted in over 25-fold higher levels of ALS messenger RNA in some transformants as compared with those expressing the native genes. However, ALS specific activity increased at most twofold, indicating that the amount of functional enzyme and messenger RNA are not correlated.

SUMMARYFructans are linear or brancbed polymers containing a single sucrose and repeating fructose residues. An early model for fructan biosyntbesis in bigber plants suggested that partial synthesis of tbe polymer occurred in tbe cell cytosol. Tbe current model suggests tbat synthesis requires the interaction of two separate fructosyltransferases located in the vacuole. Tobacco lines containing a chemically induced promoter, directing expression of the Bacillus amyloliquefaciens SacB gene in tbe present study, provided an opportunity to regulate and target fructan synthesis to tbe cytosol of transgenic plants. Induced expression of tbe gene led to rapid destruction of leaf tissue. Amino acid substitution at a bigbly conserved site (Arg^^^) in tbe SacB gene reduced tbe fructosyltransferase efficiency witbout reducing tbe invertase activity of tbe enzyme. Expression of tbe mutant gene in transgenic tobacco also resulted in leaf damage. Hov^^ever, the appearance of necrotic tissue was greatly delayed. The results suggest tbat tbe pbenotype is due to accumulation of fructan in tbe cytosol. Fructan metabolism in tbe cytosol of potato tubers was also detrimental to development. Tuber size and starcb synthesis was significantly reduced in lines containing tbe untargeted gene. Transgenic tobacco and potato containing tbe SacB gene offer an opportunity to study tbe metabolism of fructan and the effect of accumulation on plant cell development.

Fructans are linear or brancbed polymers containing a single sucrose and repeating fructose residues. An early model for fructan biosyntbesis in bigber plants suggested that partial synthesis of tbe polymer occurred in tbe cell cytosol. Tbe current model suggests tbat synthesis requires the interaction of two separate fructosyltransferases located in the vacuole. Tobacco lines containing a chemically induced promoter, directing expression of the Bacillus amyloliquefaciens SacB gene in tbe present study, provided an opportunity to regulate and target fructan synthesis to tbe cytosol of transgenic plants. Induced expression of tbe gene led to rapid destruction of leaf tissue. Amino acid substitution at a bigbly conserved site (Arg^^^) in tbe SacB gene reduced tbe fructosyltransferase efficiency witbout reducing tbe invertase activity of tbe enzyme. Expression of tbe mutant gene in transgenic tobacco also resulted in leaf damage. Hov^^ever, the appearance of necrotic tissue was greatly delayed. The results suggest tbat tbe pbenotype is due to accumulation of fructan in tbe cytosol. Fructan metabolism in tbe cytosol of potato tubers was also detrimental to development. Tuber size and starcb synthesis was significantly reduced in lines containing tbe untargeted gene. Transgenic tobacco and potato containing tbe SacB gene offer an opportunity to study tbe metabolism of fructan and the effect of accumulation on plant cell development.