Promoter methylation and progressive transgene inactivation inArabidopsis

Agrobacterium-transformedArabidopsis plants were generated and the stability of their T-DNA-encoded resistance to kanamycin was examined. Of seven families, each homozygous for a single insertion event, two showed progressive inactivation of resistance over four generations of inbreeding. Loss of resistance was associated with methylation of anSst II site in thenos promoter of the kanamycin resistance gene. Treatment of plant roots from inactive lines with the demethylating agent 5-azacytidine restored the ability of such lines to form callus on kanamycin-containing media. These observations are consistent with the view that methylation is a factor in the progressive inactivation of transgenes inArabidopsis.     

Agrobacterium-mediated transformation of Asparagus officinalis L.: molecular and genetic analysis of transgenic plants

Four long-term embryogenic lines of Asparagus officinalis were co-cultured with the hypervirulent Agrobacterium tumefaciens strain AGL1Gin carrying a uidA gene and an nptII gene. 233 embryogenic lines showing kanamycin resistance and -glucuronidase (GUS) activity were obtained. Transformation frequencies ranged from 0.8 to 12.8 transformants per gram of inoculated somatic embryos, depending on the line. Southern analysis showed that usually 1 to 4 T-DNA copies were integrated. Regenerated plants generally exhibited the same insertion pattern as the corresponding transformed embryogenic line. T₁ progeny were obtained from crosses between 6 transformed plants containing 3 or 4 T-DNA copies and untransformed plants. They were analysed for GUS activity and kanamycin resistance. In three progenies, Mendelian 1:1 segregations were observed, corresponding to one functional locus in the parent transgenic plants. Southern analysis confirmed that T-DNA copies were inserted at the same locus. Non-Mendelian segregations were observed in the other three progenies. T₂ progeny also exhibited non-Mendelian segregations. Southern analysis showed that GUS-negative and kanamycin-sensitive plants did not contain any T-DNA, and therefore inactivation of transgene expression could not be responsible for the abnormal segregations.     

Stress and domestication traits increase the relative fitness of crop-wild hybrids in sunflower

After a decade of transgenic crop production, the dynamics of gene introgression into wild relatives remain unclear. Taking an ecological genetics approach to investigating fitness in crop-wild hybrid zones, we uncovered both conditions and characteristics that may promote introgression. We compared diverse crop-wild hybrid genotypes relative to wild Helianthus annuus under one benign and three stressful agricultural environments. Whereas relative fitness of crop-wild hybrids averaged 0.25 under benign conditions, with herbicide application or competition it reached 0.45 and was more variable. In some instances, hybrid fitness matched wild fitness (approximately 1). Thus, wild populations under agronomic stress may be more susceptible to introgression. Although 'domestication' traits are typically considered unlikely to persist in wild populations, we found some (e.g. rapid growth and early flowering) that may enhance hybrid fitness, especially in stressful environments. Rigorous assessment of how particular genotypes, phenotypes, and environments affect introgression will improve risk assessment for transgenic crops.     

Lifespan and stress resistance of Caenorhabditis elegans are increased by expression of glutathione transferases capable of metabolizing the lipid peroxidation product 4-hydroxynonenal

Caenorhabditis elegans expresses a glutathione transferase (GST) belonging to the Pi class, for which we propose the name CeGSTP2-2. CeGSTP2-2 (the product of the gst-10 gene) has the ability to conjugate the lipid peroxidation product 4-hydroxynonenal (4-HNE). Transgenic C. elegans strains were generated in which the 5'-flanking region and promoter of gst-10 were placed upstream of gst-10 and mGsta4 cDNAs, respectively. mGsta4 encodes the murine mGSTA4-4, an enzyme with particularly high catalytic efficiency for 4-HNE. The localization of both transgenes was similar to that of native CeGSTP2-2. The 4-HNE-conjugating activity in worm lysates increased in the order: control相似文献   

Germline transgenesis of the ascidian Ciona intestinalis by electroporation

Microinjection of the Minos transposon is the only reported technique for generating stable transgenic lines in the cosmopolitan ascidian, Ciona intestinalis. To establish a more amenable method for generating stable transgenic Ciona, we examined the possibility of using electroporation of DNA into eggs. From 0-44.4% of electroporated individuals transmitted transgenes to the next generation. The transgene was integrated into one chromosome and multiple copies of the transgene were inserted into one site of the chromosome, indicating that electroporation is an easy and powerful technique for achieving stable transgenesis in C. intestinalis. Together with possible inland culture of this ascidian, this technique will be useful for generating stable lines which have reporter gene expression in a specific tissue or organ and the generation of transposase-expressing stable transgenic (jump-starter) lines and mutator lines which contain a lot of Minos transposons in an insertion position.     

Strategy of Production and Utilization of Immortal Cells

The current methods of production of conditionally immortal cells in vivo and in vitro have been considered, including the method based on transgenesis of animals. Examples are given for utilization of conditionally immortal cells obtained in vivo from tissues of transgenic mice and rats carrying the gene of mutant T-antigen tsA58 SV40. The recent studies were analyzed, which concern the investigation and utilization of embryonic and regional stem cells, as well as immortal cells obtained through transfection of the recombinant construct of telomerase gene into human cells. The main problems of cell biotechnology are discussed.     

The Problems of Specific Phytoimmunity

Recent studies established that the specificity of phytoimmunity is shaped at the initial stages of genic interactions between a pathogen and its plant host. While elicitors, the products of pathogen avirulence genes (avr-genes), have been already investigated in great detail, there are few studies on the products of plant resistance genes (R-genes) recognizing these elicitors. This review deals with examples of nonspecific and specific elicitors of some R-genes as well as the systems that transduce the elicited signals to the plant genome and evoke immune responses in the plant cells. Several types of immunosuppressors characteristic of pathogens are considered. The molecular-genetic studies of the relations between the pathogens and their plant hosts supplement the already existing arsenal for plant protection with new technologies, such as the genetically engineered plant resistance and the resistance induced by biogenic elicitors.     

PCR rescue and analysis of transgene sequences directly from crude extracts of transgenic embryos and plants

We report the application of a PCR-based method in conjunction with automated sequencing for the reliable detection and verification of transgenes in crude extracts of leaf and callus tissue from different plant species. Transformed tissue can be identified easily at any stage of the regeneration process, whether it is via embryogenesis or organogenesis. This allows researchers to focus their attention and resources on truly transformed tissues and avoid unwittingly culturing untransformed tissues. This protocol can also be used to rescue relatively large PCR products as well as duplexing the detection of transgenes. Direct sequencing of the PCR products allows confirmation of the integrity of the transgenein planta.