Non-cytotoxic PAP mutants
a technology of pap mutants and cytotoxic substances, which is applied in the direction of dna/rna fragmentation, plant/algae/fungi/lichens ingredients, fungi, etc., to achieve the effect of effectively interfering with the ability of a cell to manufacture proteins
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example 1
[0067]Induction of PAP expression in yeast is known to have a cytotoxic effect (1). This cytotoxicity was presumed to be a result of translation inhibition due to depurinated rRNA. This example provides evidence that PAP depurination can occur in the absence of cytotoxicity and that both events are independent. It also describes a nontoxic mutant form of PAP that retains full antiviral activity and is expressed to significantly high levels in both plants and yeast host systems. The mutants were isolated by engineering several mutations into specific regions of the PAP gene. Following the mutagenesis, the resulting clones were screened for toxicity, viability, as well as depurination activity in yeast. Several of these isolated mutants (as shown in Table 1) were found to depurinate ribosomes in the absence of toxicity. One of these isolated proteins was studied in tobacco plants to demonstrate the presence and magnitude of antiviral activity.
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Construction of the Mutant PAPY123...
example 2
[0088]PAPY123A does not autoregulate the accumulation of its own mRNA To measure the effect of PAPwt and PAPY123A on accumulation of its mRNA, Yeast cells, containing NT242 were harvested at various times after induction by galactose, and the level of PAP mRNA was measured by RNase protection assay. A 252 nt [32P]-labeled minus-strand RNA corresponding to the 3′ end of PAP mRNA was transcribed and hybridized in the presence of excess probe with total RNA extracted from cells harboring PAPwt and PAPY123A plasmids. A 281 nt [32P]-labeled minus-strand CYH2 RNA, which encodes the constitutively expressed ribosomal protein L29, served as the internal loading control (Fried et al., Nucleic Acids Res. 10:3133-3148 (1982)). Samples were electrophoretically separated and the intensities of the protected bands were quantified using a PHOSPHORIMAGER. The ratios for signals from the PAPwt or PAPY123A mRNAs to the CYH2 mRNA were used as relative measures of the steady-state abundance of the PAPw...
example 3
[0089]To examine the relationship between ribosome depurination and mRNA destabilization in cells expressing PAP, a highly sensitive primer extension analysis was used to examine the extent of ribosome depurination and mRNA turnover at different times after induction of PAP expression. PAP mRNA levels were quantified by RNAse protection analysis using the U3 RNA as the internal control. Quantification of the level of depurination in the RNA samples from wild type PAP (NT188) and L71R PAP (NT538) indicated that depurination was detected two hours after PAP induction and maximal depurination of rRNA in the cell occurred by between 2-4 hours after induction (FIG. 4). RNase protection analysis of mRNA levels indicated that wild type PAP mRNA levels increased up to four hours on galactose (FIG. 5B). PAP mRNA was destabilized after 4 hours even when transcription was not repressed, indicating that the rate of RNA degradation exceeded the rate of RNA synthesis (FIG. 5B). Ribosome depurinat...
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