Objectives: Non-viral methods of gene transfer have been preferred in gene therapy approaches for several reasons, particularly for their safety, simplicity and convenience in introducing heterologous DNA into cells. Polyomavirus virus-like particles (VLPs) represent a promising carrier for encapsidation of foreign nucleic acids for gene therapy. For the development of such gene delivery systems as well as for providing reagents for improving virus diagnostics, an efficient yeast expression system for the generation of different polyomavirus VLPs was established. Methods: A galactose-inducible Saccharomyces cerevisiae yeast expression system was used. Formation of empty VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Cross-reactivity of the major capsid proteins (VP1) of different polyomaviruses was analyzed by Western blot using rabbit and mice sera raised against the VP1 proteins. Results: VP1 of polyomaviruses from humans (JC polyomavirus and serotypes AS and SB of BK polyomavirus), rhesus monkeys (simian virus 40), hamsters (hamster polyomavirus), mice (murine polyomavirus) and birds (budgerigar fledgling disease virus) were expressed at high levels in yeast. Empty VLPs formed by all yeast-expressed VP1 proteins were dissociated into pentamers and reassociated into VLPs by defined ion and pH conditions. Different patterns of cross-reactivity of the VP1 proteins with heterologous mice and rabbit sera were observed. Conclusion: The developed heterologous yeast expression system is suitable for high-level production of polyomavirus VLPs. Yeast-derived VLPs are generally free of toxins, host cell DNA and proteins. These VLPs might be useful for the generation of new diagnostical tools, gene delivery systems and antiviral vaccines.
Early auxin-regulated tobacco cDNAs, belonging to the Aux/IAA gene family have been isolated by screening of a cDNA library prepared from auxin-treated suspension-grown etiolated seedlings of Nicotiana tabacum. The probes used were either RT-PCR fragments or an insert resulting from mRNA differential display selection. All of them possessed the structural features which characterize the Aux/IAA gene products. The auxin response of three distinct Nt-iaa subclasses has been characterized in terms of kinetics, dose-response and specificity as several plant hormones and chemicals have been tested for their ability to alter Nt-iaa mRNA accumulation. Differences of auxin responses have been observed between the Nt-iaa analysed, revealing significant differences of regulation. The effect of the protein synthesis inhibitor cycloheximide suggested that Nt-iaa2.3, Nt-iaa4.3 and strictly related genes can be classified as primary auxin-responsive genes and Nt-iaa28 as a late one. The steady-state mRNA level of these Nt-iaa has also been determined in organs of tobacco plants.
Probes corresponding to the auxin‐inducible genes parA and parB, as well as two aux genes, here denoted Nt‐aux8 and Nt‐aux16, were amplified from tobacco (Nicotiana tabacum L.) cDNA. Transcript levels of these genes were analysed in wild‐type tobacco plants and in the transgenic IAA‐overproducing line C, and related to the endogenous IAA level. In addition, effects of naphtyl‐1‐acetic acid (NAA) treatment on the expression of these genes were determined in both genotypes. Two separate situations were identified. Nt‐aux8 and Nt‐aux16 steady‐state mRNA levels were positively correlated with the IAA content in wild‐type and line C plants. Addition of NAA induced these two genes strongly in wild‐type plants, but only slightly in line C plants. On the other hand, mRNA levels of the parA and parB genes varied more with the organ analysed and its age than with the IAA level, and were induced by NAA treatment to a similar extent in the two genotypes. The results suggest that expression of the two Nt‐aux genes is closely related to the endogenous auxin level, whereas expression of the parA and parB genes to a greater extent is influenced by additional factors.
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