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Evolutionary conservation and DNA binding properties of the Ssh7 proteins from Sulfolobus shibatae

Science China(Life Sciences) 2002年第6期45卷 583-592页

作者：陈绪林郭荣黄力 Ray Hong Pomona College CA91711

The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DMA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the c/s-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both native and recombinant forms of the protein behaved in a similar fashion in the assays, suggesting that the interaction of Ssh7 with DNA is not affected either by specific lysine methylation found in the native Ssh7 proteins or by the difference between the two Ssh7 isomers in amino acid sequence. Our data show that Ssh7 binds duplex DNA fragments with a binding size of - 6.6 base pairs and an apparent dissociation constant of (0.7-1.0)×10-7 mol/L under the assay conditions employed in the present study. In addition, Ssh7 binds more tightly to negatively supercoiled DNA than to linear or relaxed DNA.

关键词： hyperthermophilic archaea, DNA binding proteins, ssh7a/ssh7b genes, protein-DNA interaction.

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The toxicity test and hypothetical model of Bacillus thuringiensis Cry1Aa helix4

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Science China(Life Sciences) 2002年第6期45卷 561-568页

作者：苏彦辉罗静初朱玉贤曲红张杰 Vachon Vincent Laprade Raynald Groupe de recheche en transport membranaire Universite de MontrealMontrealQuebecCanada

Development of targeted biological agents against agricultural insect pests is of prime importance for the elaboration and implementation of integrated pest management strategies that are environment-friendly, respectful of bio-diversity and safer to human health through reduced use of chemical pesticides. A major goal to understand how Bt toxins work is to elucidate the functions of their three domains. Domains II and III are involved in binding specificity and structural integrity, but the function of Domain I remains poorly understood. Using a Manduca sexta BBMV (brush border membrane vesicles) system, we analyzed its responses to Cry1Aa 15 single-point mutations with altered Domain I helix 4 residues. Light scattering assay showed that toxicity was almost lost in 3 mutants, and we observed significantly reduced toxicity in other 7 mutants. However, 5 mutants retained wild-type toxicity. Using computer software, we simulated the three-dimensional structures of helix 4. Both experimental and bioinformatic analysis showed that residues in Cry1Aa Domain I helix 4 were involved in the formation of ion channels that is critical for its insect toxicity.

关键词： Bt toxin proteins, light scattered assay, ion channel, molecular model.

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