Evaluation of copper-inducible fungal laccase promoter in foreign gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The promoter region of copper-inducible laccase gene, LCC1, from Pycnoporus coccineus was explored in the heterologous expression of foreign protein in Pichia pastoris. The promoter region (P_PPLCC1) was isolated and used to replace the methanol-inducible AOXI promoter (P_AOX1) of pPICHOLI-2, an episomal expression vector for P. pastoris, to generate a new copper-inducible expression vector. The promoter activity of P_PPLCC1 was compared with those of P_AOX1 and P_CUP1, a copper-inducible promoter of a commercial vector pPICHOLI-C, using a laccase gene as a reporter gene in P. pastoris GS115. Reporter laccase activity of the culture broth reached 182 and 43 units/L for P_PPLCC1 and P_CUP1, respectively, after induction with 0.2 mM CuSO₄ at OD₆₀₀ = 1 and culture for 120 h at 15°C in complex medium containing 1% glucose. For P_AOX1 activity, yeast cells harboring P_AOX1-laccase plasmid were cultured for 120 h at 15°C in complex medium with intermittent feeding with 1% methanol every 12 h to avoid methanol toxicity. Laccase activity of culture broth was 124 units/L. Conclusively, P_PPLCC1 is a new copper-inducible promoter that shows superior performance in terms of efficiency of laccase production compared to commercial vectors. P_PPLCC1 is additionally superior to P_AOX1 since it does not require laborious feeding with a carbon source.     

Progesterone-dependent and -independent expression of the multidrug resistance type I gene in porcine granulosa cells

A primary role of plasma membrane P-glycoprotein (P-gp), encoded by multidrug resistance type I (MDR1), is to protect against naturally occurring xenotoxics. Progesterone (P₄) profoundly influences MDR1 expression in granulosa cells and luteal cells. Here, P₄ regulation of MDR1 expression was investigated in porcine granulosa cells using the P₄-mediated promoter activity assay and a P4 receptor (PR) antagonist (RU-486). The promoter activity was measured chronologically for 48 h in cells transfected with the PR response element-containing pGL3. LH could stimulate the promoter activity through endogenous P4, with a maximum activity at 5 h. MDR1 mRNA level was highly maintained at 24–36 h. Conversely, exogenous P4 prolonged the promoter activity to further 10 h, and the high level of MDR1 mRNA was maintained even at 48 h. RU-486 completely inhibited the promoter activity, but the level of MDR1 mRNA rapidly increased in the presence of RU-486. The granulosa cells may become susceptible to RU-486 as a xenotoxic to rapidly express MDR1 for protection against it. These results indicate that MDR1 is expressed in porcine granulosa cells through P4-dependent and -independent regulations.     

Auxin-binding-protein antibodies and peptides influence stomatal opening and alter cytoplasmic pH

Previous work has shown that stomatal opening induced by indole-3-acetic acid (IAA) in epidermal strips of the orchid Paphiopedilum tonsum L. is preceded by a reduction in cytoplasmic pH (pH_i) of the guard cells. We now report that Fab fragments of an auxin-agonist antibody (D16), directed against a putative auxin-binding domain of the auxin-binding protein ABP1, induce stomatal opening and decrease guard-cell pH_i, as monitored with the acetomethoxy ester of the ratiometric pH indicator Snarf-1. Similar activity was shown by a monoclonal antibody against the same domain. The C-terminal dodecapeptide, Pz152–163 of maize ABP1 (ABPzm1) induced guard-cell alkalinization and closed stomata, as did Fab fragments of a monoclonal antibody (MAC 256) recognising the C-terminal region of ABPzm1. By implicating, for the first time, an auxin-binding protein in mediation of an auxin-dependent physiological response, these findings strongly support an auxin-receptor role for ABP1. Received: 23 December 1997 / Accepted: 16 January 1998     

苏云金芽胞杆菌dxr1基因的转录受SigH控制

【目的】萜类化合物广泛分布在生物界,是重要的生命物质。目前发现有两条萜类化合物的生物合成途径,即甲羟戊酸(MVA)途径和2-甲基-D-赤藓糖醇-4-磷酸(MEP)途径。MEP代谢途径中的关键酶1-脱氧-D-木酮糖-5-磷酸还原异构化酶(DXR,EC1.1.1.267)催化1-脱氧-D-木酮糖-5-磷酸生成MEP。枯草芽胞杆菌中dxr基因编码DXR酶,而在苏云金芽胞杆菌(Bacillusthuringiensis,Bt)中有2个基因(dxr1和dxr2)编码DXR酶。通过分析BtHD73菌株的dxr1基因的转录活性和dxr1突变体表型,明确dxr1基因的转录调控机制和功能。【方法】通过5?RACE分析dxr1的转录起始位点;β-半乳糖苷酶活性测定分析dxr1基因启动子(Pdxr1)的转录活性;采用同源重组技术分别敲除BtHD73菌株的dxr1和dxr2基因;利用总蛋白定量确定Cry1Ac蛋白产量;利用DXR检测试剂盒检测Bt菌株的DXR活性。【结果】dxr1基因的转录起始位点位于起始密码子上游39 bp处的G碱基;与出发菌株HD73相比,Pdxr1在sig H突变体中的转录活性明显降低;dxr1或dxr2基因的缺失对菌体生长、芽胞形成率和Cry1Ac蛋白产量无显著影响,但使DXR活性下降。【结论】Bt中dxr1基因的转录受Sig H控制,dxr1基因的缺失影响DXR的活性。