Molecular screening of interleukin-6 gene promoter and influence of −174G/C polymorphism on breast cancer

Interleukin-6 (IL-6) is a cytokine involved in different physiologic and pathophysiologic processes including carcinogenesis. In 2003, a single nucleotide polymorphism (−174G/C) of the IL-6 gene promoter has been linked to breast cancer prognosis in node-positive (N+) breast cancer patients. Since, different studies have led to conflicting conclusions about its role as a prognostic and/or diagnostic marker. The primary aim of our study was to investigate the link between −174G/C polymorphism and breast cancer risk on the one hand, and −174G/C polymorphism and prognosis in different groups of patients: sporadic N+ breast cancers (n = 138), sporadic N− breast cancers (n = 95) and familial breast cancer (n = 60) on the other hand. The variables of interest were disease-free survival and overall survival. The secondary aim of the study was to screen IL-6 gene promoter using direct sequencing to identify new polymorphisms in our French Caucasian breast cancer population. No association or trend of association between −174G/C polymorphism of IL-6 gene promoter gene and breast cancer diagnosis or prognosis was shown, even in meta-analyses. Furthermore, we have identified four novel polymorphic sites in the IL-6 gene promoter region: −764G → A, −757C → T, −233T → A, 15C → A.     

Analysis of the activity of a γ-gliadin promoter in transgenic wheat and characterization of gliadin synthesis in wheat by MALDI-TOF during grain development

The wheat grain is the most important organ for human food and therefore is the target for much research focused on modifying its composition to improve nutritional and functional components. Genetic transformation provides a precise tool to alter the composition of wheat grain by expressing new genes or by down-regulating groups of proteins encoded by multigene families such as gliadins, which contain clusters of epitopes that are active groups in triggering celiac disease. For such work, specific promoters are required to express such constructs in the wheat endosperm. In the present study we report the isolation and characterization of a γ-gliadin promoter from transgenic wheat, and the analysis of gliadin synthesis during grain development in bread wheat by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI/TOF MS). The γ-gliadin promoter fragment was isolated from bread wheat by genome walking and was re-introduced, driving the expression of the gusA gene, by particle bombardment, giving fifteen independent transgenic lines. Detailed analysis of the sequence of the 885 bp promoter fragment showed that it contains three prolamin boxes but only one is conserved according to the consensus sequence reported. The AACA/TA motif is present twice in published γ-gliadin promoter sequences. The RY element i.e., CATGCAT or CATGCAC, is also present twice in the published promoter. Transgenic lines were classified as high, medium, and low expressers. The expression of the gusA gene was found only in the seeds of the transgenic lines. GUS staining was first detected in the outer endosperm of the lobes, and then it extended to the whole outer endosperm. GUS staining was not found in the aleurone layer nor in the embryo. The qRT-PCR data confirmed the data obtained by GUS staining. The expression of the gusA gene determined by qRT-PCR for the high expresser line (B281) was 4 and 8 times higher than that of medium (B282) and low (B286) expresser lines, respectively. MALDI/TOF-MS showed that gliadins exhibited different patterns of synthesis during the course of seed maturation. Thus, gliadins with masses higher than 36,000 Da were synthesised within the first 12 days post anthesis while those with masses lower than 36,000 Da were synthesised later. Results of GUS staining, qRT-PCR and MALDI/TOF-MS showed that the γ-gliadin promoter reported in this work could be a good candidate to downregulate wheat gliadins.     

Recombinant Candida rugosa LIP2 expression in Pichia pastoris under the control of the AOX1 promoter

The LIP2 isoenzyme gene from Candida rugosa has been completely synthesised and functionally expressed under the AOX1 promoter control in Pichia pastoris. The on-line monitoring and control of methanol, the key inducer carbon source in fed-batch cultures, has enhanced the yield product/biomass 7.8-fold and the productivity 12.8-fold compared to the best batch cultivation with the Pichia system and, 10-fold compared to the fed-batch cultivation process using the native C. rugosa strain.Nevertheless, the high ionic strength of culture broth favoured aggregation of Lip2, leading to total loss of lipolytic activity. After cultivation, a diaultrafiltration process was implemented to diminish ionic strength, allowing for the recovery of lipolytic activity in the diaultrafiltrate. The developed bioprocess resulted into a reproducible product in terms of quality and productivity.     

Improvement of isoflavone aglycones production using β-glucosidase secretory produced in recombinant Aspergillus oryzae

β-Glucosidase (BGL1) from Aspergillus oryzae was efficiently produced in recombinant A. oryzae using sodM promoter-mediated expression system. The yield of BGL1 was 960 mg/l in liquid culture, which is 20-fold higher than the yield of BGL1 produced using the yeast Saccharomyces cerevisiae. Recombinant BGL1 converted isoflavone glycosides into isoflavone aglycones more efficiently than β-glucosidase from almond. In addition, BGL1 produced isoflavone aglycones even in the presence of the insoluble form of isoflavone glycosides.     

Developmental regulation of the composite CAG promoter activity in the murine T lymphocyte cell lineage

Promoter selection is of utmost importance for the study of in vivo gene function using transgenic models. In the present study, we have analyzed the expression of the GFP marker under the control of the composite CAG promoter in the lymphoid compartment of several transgenic mouse strains. Despite the ability of the CAG promoter to drive gene expression in almost all tissues examined to date, its activity appears to be developmentally regulated within the T lymphocyte cell lineage. In particular, CD4 and CD8‐expressing, thymic immature T cells displayed lower levels of the GFP marker when compared with both bone marrow precursors and mature circulating T cells, suggesting a transient downregulation of CAG activity during T cell development. Alternative promoters may therefore be preferred for the study of T cell development in vivo using a transgenic approach. genesis 47:799–804, 2009. © 2009 Wiley‐Liss, Inc.     

NADPH缺陷型Corynebacterium glutamicum重组菌株的构建及其性能分析

[目的]改造谷氨酸棒杆菌(Corynebacterium glutamicum)中NADPH合成途径,阻断胞内NADPH的合成,获得1株NADPH营养缺陷型菌株。[方法]通过失活L-赖氨酸高产菌C. glutamicum Lys-χ中葡萄糖-6-磷酸脱氢酶(Zwf)和苹果酸酶(MalE)并将NADP~+依赖型异柠檬酸脱氢酶(NADP~+-Icdcg)替换成变形链球菌(Streptococcus mutans)中的NAD~+-Icdsm,阻断胞内NADPH的合成。随后结合辅因子工程,引入大肠杆菌(Escherichia coli)中膜结合吡啶核苷酸转氢酶(PntAB)并通过不同强度启动子控制PntAB的表达水平。最后,分析不同重组菌中胞内氧化还原水平和L-赖氨酸生产强度的变化。[结果]重组菌C.glutamicum Lys-χΔZMI_(Cg)::I_(Sm)(即Lys-x1)胞内检测不到NADPH,为1株NADPH营养缺陷型菌株。该重组菌只在以葡萄糖酸为碳源的基础培养基中生长和积累L-赖氨酸,而以葡萄糖、丙酮酸、α-酮戊二酸和草酰乙酸为碳源时无法生长。此外,表达E.coli中的PntAB可回补重组菌Lys-χ1胞内NADPH的水平,但由于不同强度启动子控制PntAB表达水平不同,重组菌胞内NADPH水平也不同,并影响L-赖氨酸的生产强度。[结论]重组菌Lys-χ1可作为有效的底盘细胞,用于考察不同的NADPH再生策略,获得不同胞内NADPH水平的重组菌株,为进一步阐明NADPH调控微生物细胞生理代谢功能的机制提供研究基础。     

琥珀蚕孵化酶基因AaHE的克隆及启动子活性分析

【目的】琥珀蚕Antheraea assama具有典型的野蚕特征,蚕卵孵化不齐,严重影响琥珀蚕的室内规模化饲养。本研究旨在探究对琥珀蚕卵孵化起关键作用的孵化酶(hatching enzyme)基因及其启动子序列特征,为进一步选择合适的抑制剂或促进剂调节琥珀蚕卵的孵化奠定基础。【方法】采用RACE技术克隆琥珀蚕孵化酶基因的cDNA全长序列,对基因序列进行生物信息学分析;采用qRT-PCR检测琥珀蚕孵化酶基因在琥珀蚕不同发育天数卵中及5龄第3和4天幼虫不同组织(丝腺、马氏管、头、中肠、脂肪体、表皮、血液、精巢和卵巢)中的表达情况;采用染色体步移克隆琥珀蚕孵化酶基因的启动子序列,构建昆虫细胞重组表达载体转染家蚕Bombyx mori BmN细胞,检测琥珀蚕孵化酶基因启动子活性。【结果】获得了琥珀蚕孵化酶基因AaHE(GenBank登录号： KT336227.1)全长cDNA序列,长993 bp,编码294个氨基酸,预测蛋白质分子质量为33.7 kD,理论等电点为5.17。AaHE氨基酸序列含有一个信号肽和一个ZnMc结构域,AaHE是一种含有HExxH锌结合位点的锌依赖性蛋白水解酶,该类酶既是肽酶,同时又是一种消化酶。AaHE在琥珀蚕孵化前的卵及5龄幼虫中肠中特异性高表达,分别与AaHE的肽酶和消化酶的属性相吻合。AaHE启动子核心区存在多个转录因子结合位点,这可能与转录因子参与调节AaHE的表达有关。启动子活性分析表明,AaHE启动子在家蚕BmN细胞中能够启动EGFP基因的表达,具有明显的启动子活性。【结论】AaHE是锌依赖性蛋白水解酶,其启动子核心区存在多个转录因子结合位点。本研究为选择合适的抑制剂或促进剂调节琥珀蚕卵的孵化率提供了参考。     

茶菊品种‘14-C-1’的CmF3H基因及启动子克隆与表达分析

该研究以自育茶菊品种‘14-C-1’为材料,克隆了一个黄烷酮3-羟化酶(F3H)基因,命名为CmF3H。生物信息学分析表明,‘14-C-1’CmF3H的cDNA序列(GenBank登录号MW454869)全长为1284 bp,开放阅读框为1095 bp,编码364个氨基酸,编码蛋白的理论分子量为41.19kD,等电点为5.57,不稳定系数为39.51,平均亲水性-0.465,脂肪系数为83.02。氨基酸序列分析表明,‘14-C-1’CmF3H蛋白属于2-酮戊二酸依赖双加氧酶(2-ODD)蛋白家族,具有2-酮戊二酸双加氧酶结构域。系统发育分析结果显示,‘14-C-1’与菊花栽培种‘SU07’处于同一进化节点上,二者亲缘关系最近。采用染色体步移方法克隆了‘14-C-1’CmF3H启动子序列(GenBank登录号MW463894),全长1217 bp,启动序列分析发现其含有光响应元件、干旱和ABA响应元件、MYB识别和结合位点和组织器官发育元件等。实时荧光定量PCR分析表明,CmF3H在‘14-C-1’的根、茎、叶、花蕾、舌状花和筒状花等不同组织部位均有表达,在筒状花中表达量最高,其次为茎、叶、花蕾、舌状花,根中表达量最低。该研究结果为进一步揭示菊花黄酮类化合物的生物合成机制奠定了基础。     

染色质调节元件与不同启动子的相互作用对基因表达调控的影响

为探究DNA序列元件对不同启动子调节转基因稳定表达的影响,利用遍在染色质开放元件 (Ubiquitous chromatin opening elements,UCOE) 和基质黏附序列 (Scaffold/matrix-attachment regions,MAR) 分别与含增强子的oct4基因启动子、含CpG岛的sox2基因启动子和不含调控元件的nanog基因启动子以及同时包含增强子和CpG岛的CMV启动子组合构建pOCT4-MAR、pOCT4-UCOE、pSOX2-MAR、pSOX2-UCOE、pNANOG-MAR、pNANOG-UCOE、pCMV-UCOE、pCMV-MAR等质粒,分析这些质粒稳定转染后的表达量和嵌合表达差异。结果发现,UCOE与含增强子元件的oct4启动子组合能较稳定高效表达,而MAR与含CpG岛的sox2启动子组合能较稳定高效表达。利用排除位置效应原因的嵌合表达对染色质高级结构调控基因表达的稳定性分析表明：(1) 通常情况下UCOE比MAR调节的表达载体的表达更高效和更稳定;UCOE连接含CpG岛的启动子形成开放染色质调节的高表达更稳定;(2) MAR与启动子上TATA盒或增强子可能通过染色质环产生高表达,但相对不稳定。结论：染色质调节元件UCOE和MAR与启动子调控元件之间能通过染色质开放状态或染色质环调控基因稳定表达。