Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.

The thymidylate synthase (TS) gene is expressed at much higher levels in proliferating cells than in quiescent cells. We have been studying the sequences that are important for regulating the mouse TS gene. We previously showed that DNA sequences upstream of the essential promoter elements as well as downstream of the ATG codon are both necessary (but neither is sufficient) for normal regulation in growth-stimulated cells. In the present study, we examined the possible roles of the coding region, polyadenylation signal, and introns as downstream regulatory elements. Minigenes consisting of 1 kb of the TS 5'-flanking region, the coding region (with or without various introns at their normal locations), and polyadenylation signals from the TS gene, the human beta-globin gene, and the bovine growth hormone gene were stably transfected into wild-type mouse 3T6 cells. Minigenes that contained introns 5 and 6, 1 and 2, or 1 alone were regulated regardless of which polyadenylation signal was included. A minigene that contained an internally deleted version of intron 1 was also regulated in response to growth stimulation. However, when all introns were omitted, there was little if any change in the level of minigene expression as cells progressed from G1 through S phase. These observations indicate that TS introns contain sequences that are necessary for normal growth-regulated expression of the mouse TS gene. These sequences appear to be associated with sequences that are important for splicing and to function in cooperation with upstream regulatory elements to bring about normal S-phase-specific expression.     

Stimulation of gene expression by introns: conversion of an inhibitory intron to a stimulatory intron by alteration of the splice donor sequence.

Efficient expression of many mammalian genes depends on the presence of at least one intron. We previously showed that addition of almost any of the introns from the mouse thymidylate synthase (TS) gene to an intronless TS minigene led to a large increase in expression. However, addition of intron 4 led to a reduction in minigene expression. The goal of the present study was to determine why TS intron 4 was unable to stimulate expression. Insertion of intron 4 into an intron-dependent derivative of the ribosomal protein L32 gene did not lead to a significant increase in expression, suggesting that its inability to stimulate expression was due to sequences within the intron. Deleting most of the interior of intron 4, improving the putative branch point, removing purines from the pyrimidine stretch at the 3' end of the intron, or removing possible alternative splice acceptor or donor sites within the intron each had little effect on the level of expression. However, when the splice donor sequence of intron 4 was modified so that it was perfectly complementary to U1 snRNA, the modified intron 4 stimulated expression approximately 6-fold. When the splice donor site of TS intron 1 (a stimulatory intron) was changed to that of TS intron 4, the modified intron 1 was spliced very inefficiently and lost the ability to stimulate mRNA production. Our observations support the idea that introns can stimulate gene expression by a process that depends directly on the splicing reaction.     

地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增、克隆及序列测定

在地衣芽孢杆菌NCIB 6816菌株碱性蛋白酶基因已知序列的基础上,通过设计合适的引物,利用PCR(Polymerase Chain Reaction)技术从地衣芽孢杆菌2709菌株的柒色体DNA中扩增了2709碱性蛋白酶的编码序列。对两个克隆的PCR片段的全序列分析结果显示,2709碱性蛋白酶的编码序列同相应的NCIB 6816序列相比有3％左右的碱基组成差异。由此推定的2709碱性蛋白酶的氨基酸序列肯定了2709碱性蛋白酶属典型的subtilisin Carlsberg类,同时还表明来源于不同地衣芽孢杆菌菌株的subtilisin Carlsberg存在着若干氨基酸组成上的差异。     

厌氧氨氧化菌驱动脱氮途径的研究进展

厌氧氨氧化菌(anaerobic ammonia-oxidizing bacteria, AnAOB)的代谢多样性,使得该菌群能够在海洋、湿地和陆地等不同的自然生态系统中广泛分布,甚至在一些极热和极寒环境中也检测到了该菌群的存在。本文回顾并总结了厌氧氨氧化菌在不同生态系统中的发现、分布及脱氮贡献等方面的研究,分析了厌氧氨氧化菌分布的主要环境影响因素。该综述将帮助我们更好地理解全球氮循环中厌氧氨氧化菌的实际角色和功能,并基于厌氧氨氧化(anaerobicammoniaoxidation,anammox)过程,探究能与其进行协作的新型生物脱氮工艺,以期为这些工艺的研发和推广提供生态学基础和新的思考,从而实现脱氮工艺的技术变革。     

人体肠道细菌寡营养培养组条件的优化研究

【目的】探讨寡营养对人体肠道细菌培养组的条件。【方法】通过稀释富集培养基、固体平板和增菌肉汤培养基成分获得寡营养培养基。对健康人粪便样本分别用原液(0)、5、10、20、30和40倍稀释的富集培养基(添加羊血和瘤胃液的血培养瓶)连续增菌,在不同时间点(第0、3、6、9、15、27、30天)吸取增菌液,用YCFA (yeast casitone fatty acid)固体培养平板分离菌落;用YCFA增菌肉汤增菌后再次挑取单菌落,利用基质辅助激光解吸/电离飞行时间(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF)质谱和16S rRNA基因测序鉴定菌株。通过比较上述6种寡营养条件分离肠道菌群的效果,选取富集培养基原液、稀释10倍和30倍这3 种条件下分离效果较好的富集条件,与同样稀释倍数条件的固体平板和增菌肉汤分别组合成9种培养基条件,进一步优化肠道菌群的培养组条件。【结果】在6种寡营养富集培养基中,未稀释(原液)、10 倍和30倍稀释的富集培养基分离细菌的种类比其他...     

棒状腐败乳杆菌Lc7的生物学特性及益生作用

【目的】对分离自健康成人粪便样本的棒状腐败乳杆菌(Loigolactobacillus coryniformis)Lc7进行分类学鉴定和益生潜力评估。【方法】基于16SrRNA基因和基因组核心基因构建系统发育树,对Lc7进行分类学鉴定;通过耐酸和胆汁酸盐、粘附、抗氧化和抑菌实验,以及溶血、明胶酶活性和抗菌药物敏感性实验,评估Lc7的益生特性。同时,构建小鼠溃疡性结肠炎模型,评估Lc7的体内抗炎潜力。【结果】Lc7鉴定为L. coryniformis,在酸和胆汁酸盐的连续作用下,Lc7的存活率为70.17%。Lc7对HT-29细胞的粘附指数为56.33 CFU/cell,其自聚集和疏水性分别为80%和40%;Lc7对福氏志贺菌和鼠伤寒沙门菌等7个常见致病菌均有较强的抑制能力;对1,1-二苯基-2-苦基肼(1,1-diphenyl-2-picryl-hydrazyl,DPPH)和羟自由基(hydroxyl radicals,·OH)的清除率分别为91.70%和48.53%;Lc7无溶血现象和明胶酶活性,对选取的大多数抗生素均敏感。在小鼠结肠炎实验中,Lc7干预组小鼠结肠长度明显长于模型组(...     

高耐酸酒酒球菌常压室温等离子体诱变选育及其苹果酸-乳酸发酵研究

【目的】为选育出高度耐酸性酒酒球菌（Oenococcus oeni）突变菌株,研究其胁迫耐受性能及苹果酸-乳酸发酵（malolactic fermentation,MLF）能力。【方法】以酒酒球菌SD-2a为出发菌株,通过常压室温等离子体（atmospheric and room temperature plasma,ARTP）诱变技术,筛选高耐酸性酒酒球菌突变菌株,并探究其乙醇耐受性及在模拟酒和葡萄酒条件下的MLF能力。【结果】经过ARTP诱变处理后,利用pH 3.0的胁迫传代培养和分离纯化等,获得了5株β-葡萄糖苷酶活性较好的耐酸突变菌株,且在高乙醇浓度下表现出了较好的耐乙醇性。其中突变菌株ARTP-2在模拟酒中的β-葡萄糖苷酶活性和l-苹果酸累积降解量最高,且其在葡萄酒中l-苹果酸降解速率快于出发菌株,在第18天完成MLF,发酵后的葡萄酒香气成分的含量显著高于接种SD-2a的酒样。【结论】突变菌株ARTP-2具有良好的胁迫耐受性和MLF能力,对葡萄酒的香气起到积极的作用,为进一步开发优质的MLF商业发酵剂奠定基础。     

TaTPP-7A positively feedback regulates grain filling and wheat grain yield through T6P-SnRK1 signalling pathway and sugar–ABA interaction

Grain size and filling are two key determinants of grain thousand-kernel weight (TKW) and crop yield, therefore they have undergone strong selection since cereal was domesticated. Genetic dissection of the two traits will improve yield potential in crops. A quantitative trait locus significantly associated with wheat grain TKW was detected on chromosome 7AS flanked by a simple sequence repeat marker of Wmc17 in Chinese wheat 262 mini-core collection by genome-wide association study. Combined with the bulked segregant RNA-sequencing (BSR-seq) analysis of an F₂ genetic segregation population with extremely different TKW traits, a candidate trehalose-6-phosphate phosphatase gene located at 135.0 Mb (CS V1.0), designated as TaTPP-7A, was identified. This gene was specifically expressed in developing grains and strongly influenced grain filling and size. Overexpression (OE) of TaTPP-7A in wheat enhanced grain TKW and wheat yield greatly. Detailed analysis revealed that OE of TaTPP-7A significantly increased the expression levels of starch synthesis- and senescence-related genes involved in abscisic acid (ABA) and ethylene pathways. Moreover, most of the sucrose metabolism and starch regulation-related genes were potentially regulated by SnRK1. In addition, TaTPP-7A is a crucial domestication- and breeding-targeted gene and it feedback regulates sucrose lysis, flux, and utilization in the grain endosperm mainly through the T6P-SnRK1 pathway and sugar–ABA interaction. Thus, we confirmed the T6P signalling pathway as the central regulatory system for sucrose allocation and source–sink interactions in wheat grains and propose that the trehalose pathway components have great potential to increase yields in cereal crops.