Huwentoxin-I (HWTX-I) is a novel neurotoxin isolated from the venom of Orinithoctonus huwena. Based on its biological activity, HWTX-I could be developed as a pain-killer for clinical purpose. Production of HWTX-I by the bacterium or yeast expression systems resulted in poor yields and the puriWed protein was proved to have lower biological activity than that of native one. So, for the Wrst time, we introduced a new method to express HWTX-I gene in Sf9 cells using baculovirus expression system. Recombinant HWTX-I was recognized by Western blotting and then puriWed by nickel-chelating aYnity chromatography under native conditions. Recombinant HWTX-I showed identical amino acid sequence as native form and exhibited similar eVect on muscular transmission with that of native form. These results indicate that the baculovirus expression system and native puriWcation strategy are viable ways to produce active HWTX-I.

Trichomislin, a novel ribosome-inactivating protein, was cloned from the genome of Trichosanthes kirilowii Maxim. The gene was recombined to prokaryotic expression vector and the protein was puriWed by cation-exchange chromatography. The secondary structure of trichomislin was measured by circular-dichroism analysis and the ratios of α-helices and β-sheets were calculated. Trichomislin could inhibit the synthesis of protein in rabbit reticulocyte lysate systems and its reaction me hanism was to inactivate ribosome as an rRNA N-glycosidase. Antitumor analyses indicated trichomislin induced the apoptosis and inhibited the growth of choriocarcinoma cells. Further investigation showed that trichomislin could bind to and enter choriocarcinoma cells, and then increase the caspase-3 activity in a time-dependent manner. At the same time, the concentration of cytochrome c in cytosol increased while that in mitochondria decreased. These results suggested that trichomislin induced apoptosis by releasing cytochrome c from mitochondria which then triggered the caspase family member activation.

In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primerand a vector primer.However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem-loop structure and could notbescreenedout. This simplemethodproved to be efficient, reliable, inexpensive and time-saving, andmay be suitable for themolecules for which genespecific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosomewalkingandobtained 16positive results from 17 samples.

A rice PAL (phenylalanine ammonia-lyase) gene sequence (rPAL-P5), which is highly similar to and likely the same as a previously described rice ZB8PAL gene, including the 50-upstream and exon I coding regions of PAL, was isolated using PCR amplification. The expression of several PALs, including rPAL-P5, was strongly induced following inoculation with Pyricularia oryzae or treatment with a P. oryzae elicitor. To identify the promoter region induced by the P. oryzae elicitor, we constructed and subsequently transformed rPAL-P5 promoter deletion series into rice calli using particle bombardment. Results from both elicitor-inducible reporter gene and gel mobility shift assays demonstrated that the sequence -349 to -256 of the rPAL-P5 promoter includes a cis-element involved in the induction of P. oryzae.