Chromatin structure of the yeast FBP1 gene: Transcription-dependent changes in the regulatory and coding regions

We have studied the chromatin structure of the Saccharomyces cerevisiae FBP1 gene, which codes for fructose-1,6-bisphosphatase. A strong, constitutive, DNase I, micrococcal nuclease and S1 nuclease hypersensitive site is present close to the 3′ end of the coding region. In the repressed state, positioned nucleosomes exist around this site, and subtle changes occur in this nucleosomal organization upon derepression. A DNase I hypersensitive region is located within the promoter between positions ?540 and ?400 and it extends towards the gene in the derepressed state, leading to an alteration of nucleosomal positioning. Psoralen crosslinking of chromatin, which is used for the first time to study the mobility of restriction fragments from an RNA polymerase II gene, revealed that part of the promoter is nucleosome-free, in accordance with the results of DNase I digestion. A model is presented that, based on the chromatin structure, puts forward the hypothesis that the promoter UAS is located between ? 540 and ? 340. Finally, psoralen crosslinking, as well as digestions with micrococcal nuclease or restriction endonucleases suggests that most if not all of the copies of the active FBP1 gene are covered by nucleosomes.     

基于生物信息学稻米半胱氨酸蛋白酶抑制剂来源生物活性肽的虚拟筛选及分子对接研究

稻米半胱氨酸蛋白酶抑制剂(Oryzacystatin,OC)是除贮藏蛋白外一类重要的稻米蛋白,为筛选稻米OC来源的生物活性肽,以稻米OC蛋白质序列为对象进行生物信息学分析和计算机辅助酶解,建立OC蛋白虚拟模拟胃肠道消化产物的肽数据库,预测其生物活性肽序列.进一步结合分子对接虚拟筛选具有结合潜力的活性肽并探讨其与靶蛋白的...     

Analysis of the Structure of a Natural Alternating d(TA)n Sequence in Yeast Chromatin

We address here the question of the in vivo structure of a natural alternating d(TA)_n sequence found at the 3′ region of the Saccharomyces cerevisiae FBP1 gene. This sequence consists of 13 TA pairs interrupted by a TT dinucleotide in the middle of the tract. Previous experiments with cruciform-specific nucleases S1 and Endonuclease VII demonstrated the presence in vitro of a cruciform in this region. We also showed this region to be part of a nuclease hypersensitive site flanked by nucleosomes in yeast chromatin. Here we demonstrate, by means of S1 in vivo footprinting, that in yeast plasmids also adopts in vivo a non B-DNA structure which is not a cruciform. A theoretical analysis of this region shows that it contains a site susceptible to superhelical stress duplex destabilization. The locations and conditions under which alternative structures form in the wild-type sequence and in deletion mutants agree with these theoretical predictions, suggesting that some kind of denaturation is the alternative structure adopted by the sequence in vivo. This suggests that negative superhelical stress sufficient for local denaturation exists in nucleosomal DNA. We also demonstrate by micrococcal nuclease digestions that the deletion of the alternating d(TA)_n sequence modifies the chromatin hypersensitive site but does not affect nucleosome positioning. © 1997 John Wiley & Sons, Ltd.     

仿刺参胶原蛋白源二肽基肽酶抑制肽虚拟筛选及分子对接研究

目的 采用生物信息学和分子对接方法筛选仿刺参胶原蛋白来源的二肽基肽酶(dipeptidyl peptidase-Ⅳ,DPP-Ⅳ)抑制肽。方法 以仿刺参胶原蛋白序列为对象,进行生物信息学分析和计算机辅助虚拟酶解,基于生物毒性、致敏性、水溶性及吸收、分配、代谢、排泄及毒性(absorption,distribution,metabolism,excretion,toxicity,ADMET)预测,筛选得到6条具有潜在生物活性的寡肽,氨基酸序列分别为CD、CQ、CS、GR、SM、MDG,进一步通过分子对接分析肽段与DPP-Ⅳ的结合活性,并分析其结合位点及方式。结果 生物信息学分析表明仿刺参胶原蛋白是生物活性肽的潜在优良来源,经木瓜蛋白酶、碱性蛋白酶及模拟胃肠道虚拟水解后能够释放出DPP-Ⅳ抑制肽;分子对接表明俩条新肽段CS、SM通过氢键、疏水相互作用分别与DPP-Ⅳ的S1、S2活性口袋结合。结论 本研究提供了一种快速筛选刺参胶原蛋白中DPP-Ⅳ抑制肽的方法。     

Phylogenetic footprinting reveals multiple regulatory elements involved in control of the meiotic recombination gene, REC102.

REC102 is a meiosis-specific early exchange gene absolutely required for meiotic recombination in Saccharomyces cerevisiae. Sequence analysis of REC102 indicates that there are multiple potential regulatory elements in its promoter region, and a possible regulatory element in the coding region. This suggests that the regulation of REC102 may be complex and may include elements not yet reported in other meiotic genes. To identify potential cis-regulatory elements, phylogenetic footprinting analysis was used. REC102 homologues were cloned from other two Saccharomyces spp. and sequence comparison among the three species defined evolutionarily conserved elements. Deletion analysis demonstrated that the early meiotic gene regulatory element URS1 was necessary but not sufficient for proper regulation of REC102. Upstream elements, including the binding sites for Gcr1p, Yap1p, Rap1p and several novel conserved sequences, are also required for the normal regulation of REC102 as well as a Rap1p binding site located in the coding region. The data in this paper support the use of phylogenetic comparisions as a method for determining important sequences in complex promoters.     

肉源性活性肽及其计算机分析技术研究进展

肉及其副产物中含有多种蛋白质,这些蛋白质经过酶解、微生物发酵或胃肠道消化后,产生多种有益于人体的活性肽,如降血压肽（血管紧张素转化酶抑制肽）、抗氧化肽、抗菌肽和二肽基肽酶抑制肽等。肽的生物活性逐渐被人们重视,但其提取和筛选仍然受到分离鉴定技术的限制。计算机分析技术能够很好地评估及分离鉴定蛋白质作为活性肽前体蛋白的能力,模拟酶及化学作用对蛋白质的水解,预测肽序列的活性及其化学性质。本文对肉源性活性肽进行综述,并介绍计算机分析技术在分离鉴定活性肽中的应用。     

通过串联启动子实现纳豆激酶在枯草芽孢杆菌中的高效表达

本研究通过串联启动子方式实现纳豆激酶在枯草芽孢杆菌WB800中的高效分泌表达。通过对几种现有报道的强启动子的比较并对其进行串联操作,确定生产纳豆激酶的最优启动子及纳豆激酶的最高产量。本研究首先在枯草芽孢杆菌WB800中成功构建五种含不同强启动子的重组质粒p SG101(P_(HpaII)),p SG102(PBcapr E),p SG103(Plux S),p SG104(Pgsi B)和p SG105(Pyxi E),实现纳豆激酶分泌表达,并对其纤溶活性进行测定。结果表明,启动子P_(HpaII)介导的纳豆激酶纤溶活性(110.80 FU/m L)明显优于其他四种启动子。通过对启动子P_(HpaII)进行多次串联,成功构建质粒p SG106(P_(HpaII)-P_(HpaII)),p SG107(P_(HpaII)-P_(HpaII)-P_(HpaII))和p SG108(P_(HpaII)-P_(HpaII)-P_(HpaII)-P_(HpaII))。数据显示,菌株Bacillus subtilis WB800/p SG107(P_(HpaII)-P_(HpaII)-P_(HpaII))纳豆激酶产量最高为213.30 FU/m L,相比单个启动子P_(HpaII),提高了92.51%。通过对五种强启动子的比较以及对其进行串联操作,成功实现纳豆激酶在枯草芽孢杆菌WB800的高效表达,纤溶活性最高为213.30 FU/m L,与现有相关报道相比有明显优势。     

Identification of regulatory elements in the AGT1 promoter of ale and lager strains of brewer's yeast

Agt1 is an interesting α-glucoside transporter for the brewing industry, as it efficiently transports maltotriose, a sugar often remaining partly unused during beer fermentation. It has been shown that on maltose the expression level of AGT1 is much higher in ale strains than in lager strains, and that glucose represses the expression, particularly in the ale strains. In the present study the regulatory elements of the AGT1 promoter of one ale and two lager strains were identified by computational methods. Promoter regions up to 1.9 kbp upstream of the AGT1 gene were sequenced from the three brewer's yeast strains and the laboratory yeast strain CEN.PK-1D. The promoter sequence of the laboratory strain was identical to the AGT1 promoter of strain S288c of the Saccharomyces Genome Database, whereas the promoter sequences of the industrial strains diverged markedly from the S288c strain. The AGT1 promoter regions of the ale and lager strains were for the most part identical to each other, except for one 22 bp deletion and two 94 and 95 bp insertions in the ale strain. Computational analyses of promoter elements revealed that the promoter sequences contained several Mig1- and MAL-activator binding sites, as was expected. However, some of the Mig1 and MAL-activator binding sites were located on the two insertions of the ale strain, and thus offered a plausible explanation for the different expression pattern of the AGT1 gene in the ale strains. Accordingly, functional analysis of A60 ale and A15 lager strain AGT1 promoters fused to GFP (encoding the green fluorescent protein) showed a significant difference in the ability of these two promoters to drive GFP expression. Under the control of the AGT1 promoter of the ale strain the emergence of GFP was strongly induced by maltose, whereas only a low level of GFP was detected with the construct carrying the AGT1 promoter of the lager strain. Thus, the extra MAL-activator binding element, present in the AGT1 promoter of the ale strain, appears to be necessary to reach a high level of induction by maltose. Both AGT1 promoters were repressed by glucose but their derepression was different, possibly due to a distinct distribution of Mig1 elements in these two promoters.     

苦荞10 kD过敏原的重组表达及部分性质分析

以苦荞种子灌浆期cDNA文库中获得的苦荞10 kD过敏原基因序列TBAP10（tartary buckwheat 10 kD allergenprotein,TBAP10;GenBank登录号JK729379.1）为基础,构建重组表达载体pET47b-TBAP10,重组蛋白在大肠杆菌BL21 Star（DE3）中以包涵体形式表达。经包涵体复性和钴离子螯合层析纯化目的蛋白,并对其过敏活性、热稳定性及在模拟胃肠环境中的稳定性进行了分析。Western blotting显示该蛋白与苦荞16 kD过敏蛋白Fag t2存在免疫交叉反应。竞争性ELISA证明重组蛋白TBAP10具有与荞麦过敏患者血清IgE特异的结合活性;TBAP10具有强的热稳定性,能耐受15 min的沸水浴;模拟胃肠环境的消化结果显示TBAP10对胃蛋白酶具有强的耐受性,但对胰蛋白酶无耐受性。     

Chromatin-unstable boar spermatozoa have little chance of reaching oocytes in vivo

In the present study, the prevalence of chromatin instability in the fertilizing-competent sperm population in the porcine oviduct in vivo was examined through qualitative analysis of the chromatin structure status of accessory boar sperm found in in vivo-derived embryos. The binding of chromatin-unstable sperm to oviductal epithelium in vitro was also studied. To examine the sperm chromatin state, a modified fluorescence microscopic sperm chromatin structure assay was used. Among a population of 173 fertile boars, individuals were selected for according to their chromatin status: 25 animals showed more than 5% of chromatin-unstable sperm in their ejaculates, and 7 showed consistently elevated percentages of chromatin-unstable sperm in three successively collected semen samples. A positive correlation was found between incidence of chromatin instability and attached cytoplasmic droplets (r=0.44, P<0.01). Analyses of accessory spermatozoa from in vivo-derived embryos demonstrated that the proportion of chromatin-unstable sperm was significantly (P<0.05) reduced in the population of fertilizing-competent sperm in the oviduct compared with the inseminated sperm. Populations of sperm bound to the oviduct in vitro had significantly (P<0.05) lower percentages of chromatin instability than in the original diluted semen sample. In conclusion, numbers of sperm with unstable chromatin are reduced in the oviductal sperm reservoir, possibly because of associated changes in the plasma membrane that prevent sperm from binding to the oviductal epithelium. We conclude that in vivo the likelihood that sperm with unstable chromatin will reach the egg and fertilize it is low.     

Initial proteolysis of milk proteins and its effect on formation of ACE-inhibitory peptides during gastrointestinal proteolysis: a bioinformatic, in silico, approach

Angiotensin-I-converting enzyme (ACE)-inhibitory peptides are encrypted in milk protein sequences. They may express an antihypertensive effect if they are released by proteolysis in foods and/or during gastrointestinal digestion. A bioinformatic, in silico, approach was developed to evaluate how systematic initial proteolysis, i.e. cleavage after one specific type of amino acid (C-end) at a time in milk proteins, influence the formation of ACE-inhibitory peptides by subsequent gastrointestinal proteolysis. Computer simulation was done and a peptide QSAR model was used to estimate the combined ACE inhibition by digested proteins. Initial proteolytic cleavage at the C-end of amino acids isoleucine and proline gave, based on calculations, increased effect of ACE-inhibitory peptides after gastrointestinal proteolysis of milk proteins. Cleavage after most other amino acid residues had little or no effect. Results indicate that initial proteolysis in foods have to be specific in order to increase formation of bioavailable ACE-inhibitory peptides during gastrointestinal digestion.     

The multifunctional regulatory proteins ABF1 and CPF1 are involved in the formation of a nuclease-hypersensitive region in the promoter of the QCR8 gene

The abundant DNA-binding proteins ABF1 and CPF1 are members of a family of global regulators with diverse chromosomal functions in the yeast Saccharomyces cerevisiae. Recent evidence suggests that these protein factors may be involved in establishing and maintaining well-defined chromatin structures in promoter regions and other genetic elements. We have investigated the involvement of ABF1 and CPF1 in chromatin organization at the QCR8 gene, encoding subunit VIII of the mitochondrial ubiquinol-cytochrome c oxidoreductase. The promoter region of the QCR8 gene contains overlapping binding sites for ABF1 and CPF1. Nucleosome positioning studies indicate that the QCR8 gene is associated with a phased array of nucleosomes under both catabolite-repressed and derepressed growth conditions. Analysis of binding site mutants reveals that both ABF1 and CPF1 are involved in maintaining a nuclease-hypersensitive region in the QCR8 promoter. The chromatin structure at QCR8 during steady-state growth is, however, mainly dependent on binding of ABF1 to the promoter region. Implications of these findings for the role played by ABF1 and CPF1 in the regulation of mitochondrial biogenesis and other processes important for cell growth and division will be discussed.     

A new approach to species determination for yeast strains: DNA microarray-based comparative genomic hybridization using a yeast DNA microarray with 6000 genes

DNA-DNA hybridization is known as the superior method in the elucidation of relationships between closely related taxa, such as species and strain. For species determination we propose a new DNA-DNA hybridization method: the DNA microarray-based comparative genomic hybridization (CGH) method, using a yeast DNA microarray with approximately 6000 genes. The genome from a yeast strain as a sample strain (Sample) was labelled with Cy3-dye and hybridized to a single DNA microarray, together with the Cy5-labelled genome of S. cerevisiae S288C as a reference strain (Reference). The log2 ratio values [log2[Cy3(Sample)/Cy5(Reference)]: Ratio] of signal intensities of all the gene spots were estimated and divided into the following groups: Ratio < or = -1; -1 < Ratio < 1; 1 < or = Ratio. The hybridization profiles of the genomes of type strains belonging to the genus Saccharomyces were significantly different from that of S. cerevisiae S288C. The Ratio-based grouping allowed us to discriminate between some species from S. cerevisiae more clearly. Furthermore, cluster analysis discriminated between closely related species and strains. Using this method, we were able to not only perform species determination but also to obtain information on alternation in gene copy number of such gene amplifications and deletions with single-gene resolution. These observations indicated that DNA microarray-based CGH is a powerful system for species determination and comparative genome analysis.