Transient gene expression of foreign genes in preheated protoplasts: stimulation of expression of transfected genes lacking heat shock elements.

Transfection of preheated petunia protoplasts with several biologically active DNA constructs resulted in a significantly higher gene expression than that observed in transfected unheated protoplasts. It was observed with supercoiled, linearized and single-stranded DNA structures that stimulation of transient gene expression in preheated protoplasts was neither dependent on the reporter gene nor on the regulatory elements used. Heat treatment at 42 degrees C also increased expression in protoplasts transfected with a plasmid bearing the tobacco mosaic virus (TMV) translational enhancer, omega. Northern blot analysis revealed that heat treatment of protoplasts before the transfection event greatly increased the amount of the newly synthesized transcripts. Preheating of protoplasts did not affect the transfection efficiency, namely the number of transfected cells in the population, nor the amount of DNA in transfected nuclei, as was inferred from histochemical staining and Southern blot analysis, respectively. The possible mechanism by which heat treatment stimulates transient gene expression of genes lacking obvious heat shock elements is offered. The relevance of the present findings to transient gene expression in plants in general and to viral gene expression in particular is discussed.

Expression of plant genes in transfected mammalian cells: accumulation of recombinant preLHCIIb proteins within cytoplasmic inclusion bodies.

Transfection of the monkey COS-7 cells with an expression vector bearing the Lemma gibba LHCIIb AB30 or AB19 genes led to the synthesis of the LHCIIb polypeptide precursors (preLHCIIb). This was inferred mainly from Western blot analysis which has revealed the appearance of a single immunoprecipitation band following the use of three different preparations of anti-LHCIIb antibodies. Synthesis of the precursor polypeptides, not the mature processed LHCIIb protein, was evident from the molecular weight of the newly synthesized protein, inferred from its position in the gel. Expression of the AB30 and AB19 genes was also evident from the appearance of specific transcripts only in transfected cells. Immunofluorescence observations revealed the appearance of distinct fluorescent spots in about 1-2% of the transfected cells. The same was observed following immunogold staining and electron microscopy studies, which revealed a specific association of gold particles with amorphous structures only in transfected cells. The preLHCIIb synthesized by transfected COS-7 cells was insoluble in a variety of detergents and could be solubilized only by 8 M urea or 0.1 N NaOH. These properties are characteristic of proteins accumulating within inclusion bodies of prokaryotes.

Efficient functioning of plant promoters and poly(A) sites in Xenopus oocytes.

Mature Xenopus oocytes were challenged with DNA constructs including plant regulatory elements, namely, the Cauliflower mosaic virus (CaMV) 35S promoter as well as the nopaline synthase (NOS) promoter and polyadenylation signal. The bacterial chloramphenicol acetyl transferase (CAT) was used as a reporter gene. When microinjected into these cells, the plant-derived DNA constructs effectively promoted CAT synthesis in a manner dependent on the presence of the plant promoters and probably also on the polyadenylation signals. Structural studies revealed that the supercoiled structures of the above DNA plasmids were much more active in supporting CAT synthesis in microinjected oocytes than their linear forms, with clear correlation between efficient gene expression and DNA topology. In contrast, the linear forms of these plasmids were considerably more active than the supercoiled ones in transfected plant protoplasts. These findings demonstrate, for the first time, the activity of regulatory elements from plant genes in Xenopus oocytes and shed new light on the specific rules applicable for gene expression in plant and animal cells.