利用同源重组的方法，构建了具有高拷贝数和高稳定性的新型酿酒酵母表达质粒。对酵母附加体型载体pHC11进行一系列改造，得到质粒pHC11R，并用适当的酶切线性化；利用PCR反应得到人IFNa2b表达单元片段，它的两端和线性化的pHC11R的两端具有50bp左右的同源区段。上述两个片段共转化酿酒酵母，在酵母体内经同源重组后得到表达质粒pHC11R-IFNa2b，该表达质粒与pHC11-IFNoa2b相比去除了质粒上用于在大肠杆菌中复制和筛选所需的序列，在宿主菌中的稳定性和拷贝数均有明显提高，同时外源基因的表达量也获得了一定程度的提高。在此基础上，进一步构建了带有不同表达元件的pHR系列载体，使得外源基因能够通过同源重组在酿酒酵母中快速、方便地克隆和表达。

In addition to ergosterol (I), 3b, 5a, 6b-trihydroxyergosta-7, 22-diene (II), 3b-hydroxy-ergosta-5-ene (Ⅲ), 3-oxo-ergosta-4, 6, 8 (14), 22-tetraene (IV), 3b-hydroxy-5a, 8a-epidioxy-ergosta-6, 22-diene (V), 3b-hydroxy-5a,8a-epidioxy-ergosta-6, 9 (11), 22-triene (VI) and 3-oxo-ergosta-4-ene (VII), a plant hormone indole-3-acetic acid (IAA) and three new antimicrobial metabolites were characterized from the culture of Colletotrichum sp., an endophyte isolated from inside the stem of Artemisia annua. The structures of the new metabolites were elucidated by a combination of spectroscopic methods (IR, MS, 1H and 13C NMR) as 6-isoprenylindole-3-carboxylic acid (1), 3b,5a-dihydroxy-6b-acetoxy-ergosta-7, 22-diene (2) and 3b, 5a-dihydroxy-6b-phenylacetyloxy-ergosta-7, 22-diene (3), respectively. The compounds 1-3 and Ⅲ-V inhibited the growth of all the tested bacteria (Bacillus subtilis, Staphylococcus aureus, Sarcina lutea and Pseudomonas sp.) with minimal inhibitory concentrations (MICs) ranging from 25 to 75 mg: ml. Moreover, metabolites 2 and 3, together with the known sterols III and V, were inhibitory against the fungi Candida albicans and Aspergillus niger with MICs between 50 and 100 mg: ml. At 200 mg: ml, compounds 1-3, III and IV were shown to be fungistatic to the crop pathogenic fungi Gaeumannomyces graminis var. tritici, Rhizoctonia cerealis, Helminthosporium sati6um and Phytophthora capisici. This is the first report on the endophytic fungus from A. annua and the bioactive metabolites thereof.

Artemisia annua, well recognized for its production of antimalarial drug artemisinin, is seldom attacked by any of phytopathogenic fungi, which could be partially associated with the presence of endophytes. Present investigation is aiming at disclosing whether the endophytes inside A. annua produce antifungal substances. A total of 39 endophytes were isolated and fermented, and the ferment broth was evaluated in vitro for the antifungal activity against crop-threatening fungi Gaeumannomyces graminis var. tritici, Rhizoctonia cerealis, Helminthosporium sati um, Fusarium graminearum, Gerlachia ni alis and Phytophthora capsici. These plant pathogens are still causing wheat take-all, sharp eyespot, common rot, scab, snow mould, and pepper phytophthora blight, respectively. Out of 39 endophytes investigated, 21 can produce in vitro substances that are inhibitory to all or a few of the tested phytopathogens whereas the rest yielded nothing active. Moreover, the most active broth of endophyte IV403 was extracted with EtOAc and n-butanol, and comparisons of the antifungal activity of the extracts indicated that the major active metabolites were EtOAc-extractable.

The human novel gene pp5644 (GeneBank Accession No. AF289559) coding for 124 amino acids was recently cloned. Overexpression of pp5644 in Hela cells significantly inhibited the growth and colony formation. The pp5644-interacting protein FAPP1 (phosphatidylinositol-four-phosphate adaptor protein1) associated protein-1, called FASP1, was obtained by using yeast two-hybrid system. The interaction between pp5644 and FASP1 was experimentally confirmed by GST pull-down assay in vitro and co-immunoprecipitation assay in vivo. Co-localization of pp5644 and FASP1 in cytoplasm in Hela cells could further support the interaction. Based on the experimental results, it is suggested that pp5644 physically bind to FASP1 and the biological significance of this kind of interaction in vivo is discussed.

hASB-8基因是对肿瘤细胞生长具有明显抑制作用的人类新基因。其编码蛋白属于人ASB蛋白家族中的一个成员，与小鼠中的ASB-8蛋白同源性达96%。保守结构域分析显示hASB-8在N端包含4个Ankyfin re-peats，在C端包含了一个SOCS box。利用酵母双杂交技术，筛选了人的胎盘（Placenta）cDNA 文库，获得了与KASB-8相互作用的2个蛋白，Elongin C和CDK4 binding protein；并在二倍体酵母体内进行了验证。这些试验提示hASB-8蛋白可能介导肿瘤细胞中靶蛋白和泛素复合体之间的相互作用，并与肿瘤细胞靶蛋白转录调节有关。