首页 > 最新文献

BMC Molecular Biology最新文献

英文 中文
Positive cofactor 4 (PC4) contributes to the regulation of replication-dependent canonical histone gene expression 正辅因子4 (PC4)参与了复制依赖性典型组蛋白基因表达的调控
IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2018-07-27 DOI: 10.1186/s12867-018-0110-y
Aleksandra Brzek, Marlena Cichocka, Jakub Dolata, Wojciech Juzwa, Daniel Schümperli, Katarzyna Dorota Raczynska

Core canonical histones are required in the S phase of the cell cycle to pack newly synthetized DNA, therefore the expression of their genes is highly activated during DNA replication. In mammalian cells, this increment is achieved by both enhanced transcription and 3′ end processing. In this paper, we described positive cofactor 4 (PC4) as a protein that contributes to the regulation of replication-dependent histone gene expression.

We showed that PC4 influences RNA polymerase II recruitment to histone gene loci in a cell cycle-dependent manner. The most important effect was observed in S phase where PC4 knockdown leads to the elevated level of RNA polymerase II on histone genes, which corresponds to the increased total level of those gene transcripts. The opposite effect was caused by PC4 overexpression. Moreover, we found that PC4 has a negative effect on the unique 3′ end processing of histone pre-mRNAs that can be based on the interaction of PC4 with U7 snRNP and CstF64. Interestingly, this effect does not depend on the cell cycle.

We conclude that PC4 might repress RNA polymerase II recruitment and transcription of replication-dependent histone genes in order to maintain the very delicate balance between histone gene expression and DNA synthesis. It guards the cell from excess of histones in S phase. Moreover, PC4 might promote the interaction of cleavage and polyadenylation complex with histone pre-mRNAs, that might impede with the recruitment of histone cleavage complex. This in turn decreases the 3′ end processing efficiency of histone gene transcripts.

核心规范组蛋白在细胞周期的S期需要包装新合成的DNA,因此其基因的表达在DNA复制期间被高度激活。在哺乳动物细胞中,这种增加是通过增强的转录和3 '端加工来实现的。在本文中,我们将正辅因子4 (PC4)描述为一种有助于调节复制依赖性组蛋白基因表达的蛋白质。我们发现PC4以细胞周期依赖的方式影响RNA聚合酶II向组蛋白基因位点的募集。最重要的影响是在S期观察到的,PC4敲低导致组蛋白基因上RNA聚合酶II水平升高,这与这些基因转录物的总水平增加相对应。PC4过表达则产生相反的效果。此外,我们发现PC4对组蛋白前mrna独特的3 '端加工具有负面影响,这可能是基于PC4与U7 snRNP和CstF64的相互作用。有趣的是,这种效果并不依赖于细胞周期。我们得出结论,PC4可能抑制RNA聚合酶II的募集和复制依赖性组蛋白基因的转录,以维持组蛋白基因表达和DNA合成之间的微妙平衡。它在S期保护细胞免受过量组蛋白的侵害。此外,PC4可能促进裂解和聚腺苷化复合体与组蛋白前mrna的相互作用,从而阻碍组蛋白裂解复合体的募集。这反过来又降低了组蛋白基因转录本的3 '端加工效率。
{"title":"Positive cofactor 4 (PC4) contributes to the regulation of replication-dependent canonical histone gene expression","authors":"Aleksandra Brzek,&nbsp;Marlena Cichocka,&nbsp;Jakub Dolata,&nbsp;Wojciech Juzwa,&nbsp;Daniel Schümperli,&nbsp;Katarzyna Dorota Raczynska","doi":"10.1186/s12867-018-0110-y","DOIUrl":"https://doi.org/10.1186/s12867-018-0110-y","url":null,"abstract":"<p>Core canonical histones are required in the S phase of the cell cycle to pack newly synthetized DNA, therefore the expression of their genes is highly activated during DNA replication. In mammalian cells, this increment is achieved by both enhanced transcription and 3′ end processing. In this paper, we described positive cofactor 4 (PC4) as a protein that contributes to the regulation of replication-dependent histone gene expression.</p><p>We showed that PC4 influences RNA polymerase II recruitment to histone gene loci in a cell cycle-dependent manner. The most important effect was observed in S phase where PC4 knockdown leads to the elevated level of RNA polymerase II on histone genes, which corresponds to the increased total level of those gene transcripts. The opposite effect was caused by PC4 overexpression. Moreover, we found that PC4 has a negative effect on the unique 3′ end processing of histone pre-mRNAs that can be based on the interaction of PC4 with U7 snRNP and CstF64. Interestingly, this effect does not depend on the cell cycle.</p><p>We conclude that PC4 might repress RNA polymerase II recruitment and transcription of replication-dependent histone genes in order to maintain the very delicate balance between histone gene expression and DNA synthesis. It guards the cell from excess of histones in S phase. Moreover, PC4 might promote the interaction of cleavage and polyadenylation complex with histone pre-mRNAs, that might impede with the recruitment of histone cleavage complex. This in turn decreases the 3′ end processing efficiency of histone gene transcripts.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"19 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2018-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-018-0110-y","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5475741","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
Evaluation of suitable reference genes for qRT-PCR normalization in strawberry (Fragaria × ananassa) under different experimental conditions 不同实验条件下草莓(Fragaria × ananassa) qRT-PCR归一化合适内参基因的评价
IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2018-06-22 DOI: 10.1186/s12867-018-0109-4
Yunting Zhang, Xiaorui Peng, Yi Liu, Yali Li, Ya Luo, Xiaorong Wang, Haoru Tang

Strawberry has received much attention due to its nutritional value, unique flavor, and attractive appearance. The availability of the whole genome sequence and multiple transcriptome databases allows the great possibility to explore gene functions, comprehensively. Gene expression profiles of a target gene can provide clues towards the understanding of its biological function. Quantitative real-time PCR (qRT-PCR) is a preferred method for rapid quantification of gene expression. The accuracy of the results obtained by this method requires the reference genes with consistently stable expression to normalize its data.

In present study, the expression stability of seven candidate reference genes in diverse sample subsets of different tissues and fruit developmental stages, and plant subjected to light quality and low temperature treatments was evaluated using three statistical algorithms, geNorm, NormFinder, and BestKeeper. Our data indicated that the expression stability of reference genes varied under different experimental conditions. Overall, DBP, HISTH4, ACTIN1 and GAPDH expressed much more stably. PIRUV, ACTIN2 and 18S were not recommended for normalization in given experimental conditions due to low stability. In addition, the relative expression pattern of HY5 (ELONGATED HYPOCOTYL5) was conducted to further confirm the reliability of the reference genes, which demonstrated the correct adoption of reference genes was of great importance in qRT-PCR analysis.

Expression stability of reference genes from strawberry varied across selected experimental conditions. Systematic validation of reference genes prior to calculation of target gene expression level should be done to improve the accuracy and consistency of qRT-PCR analysis.

草莓因其营养价值高、风味独特、外观美观而备受关注。全基因组序列和多个转录组数据库的可用性为全面探索基因功能提供了很大的可能性。靶基因的基因表达谱可以为理解其生物学功能提供线索。定量实时PCR (qRT-PCR)是快速定量基因表达的首选方法。该方法所获得结果的准确性要求具有稳定表达的内参基因对其数据进行规范化处理。本研究采用geNorm、NormFinder和BestKeeper三种统计算法,对7个候选内参基因在不同组织、果实发育阶段和不同低温处理植物中的表达稳定性进行了评价。我们的数据表明,在不同的实验条件下,内参基因的表达稳定性有所不同。总体而言,DBP、HISTH4、ACTIN1和GAPDH的表达更加稳定。在给定的实验条件下,由于稳定性低,不建议使用PIRUV、ACTIN2和18S进行归一化。此外,通过对HY5 (ELONGATED HYPOCOTYL5)的相对表达谱进一步证实了内参基因的可靠性,说明在qRT-PCR分析中,正确选用内参基因是非常重要的。草莓内参基因在不同条件下的表达稳定性不同。为了提高qRT-PCR分析的准确性和一致性,在计算靶基因表达水平之前,应对内参基因进行系统验证。
{"title":"Evaluation of suitable reference genes for qRT-PCR normalization in strawberry (Fragaria × ananassa) under different experimental conditions","authors":"Yunting Zhang,&nbsp;Xiaorui Peng,&nbsp;Yi Liu,&nbsp;Yali Li,&nbsp;Ya Luo,&nbsp;Xiaorong Wang,&nbsp;Haoru Tang","doi":"10.1186/s12867-018-0109-4","DOIUrl":"https://doi.org/10.1186/s12867-018-0109-4","url":null,"abstract":"<p>Strawberry has received much attention due to its nutritional value, unique flavor, and attractive appearance. The availability of the whole genome sequence and multiple transcriptome databases allows the great possibility to explore gene functions, comprehensively. Gene expression profiles of a target gene can provide clues towards the understanding of its biological function. Quantitative real-time PCR (qRT-PCR) is a preferred method for rapid quantification of gene expression. The accuracy of the results obtained by this method requires the reference genes with consistently stable expression to normalize its data.</p><p>In present study, the expression stability of seven candidate reference genes in diverse sample subsets of different tissues and fruit developmental stages, and plant subjected to light quality and low temperature treatments was evaluated using three statistical algorithms, geNorm, NormFinder, and BestKeeper. Our data indicated that the expression stability of reference genes varied under different experimental conditions. Overall, <i>DBP</i>, <i>HISTH4</i>, <i>ACTIN1</i> and <i>GAPDH</i> expressed much more stably. <i>PIRUV</i>, <i>ACTIN2</i> and <i>18S</i> were not recommended for normalization in given experimental conditions due to low stability. In addition, the relative expression pattern of <i>HY5</i> (ELONGATED HYPOCOTYL5) was conducted to further confirm the reliability of the reference genes, which demonstrated the correct adoption of reference genes was of great importance in qRT-PCR analysis.</p><p>Expression stability of reference genes from strawberry varied across selected experimental conditions. Systematic validation of reference genes prior to calculation of target gene expression level should be done to improve the accuracy and consistency of qRT-PCR analysis.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"19 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2018-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-018-0109-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4859694","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 38
Laser capture microdissection for transcriptomic profiles in human skin biopsies 激光捕获显微解剖的转录组谱在人体皮肤活检
IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2018-06-19 DOI: 10.1186/s12867-018-0108-5
Silvia Santoro, Ignazio Diego Lopez, Raffaella Lombardi, Andrea Zauli, Ana Maria Osiceanu, Melissa Sorosina, Ferdinando Clarelli, Silvia Peroni, Daniele Cazzato, Margherita Marchi, Angelo Quattrini, Giancarlo Comi, Raffaele Adolfo Calogero, Giuseppe Lauria, Filippo Martinelli Boneschi

The acquisition of reliable tissue-specific RNA sequencing data from human skin biopsy represents a major advance in research. However, the complexity of the process of isolation of specific layers from fresh-frozen human specimen by laser capture microdissection, the abundant presence of skin nucleases and RNA instability remain relevant methodological challenges. We developed and optimized a protocol to extract RNA from layers of human skin biopsies and to provide satisfactory quality and amount of mRNA sequencing data.

The protocol includes steps of collection, embedding, freezing, histological coloration and relative optimization to preserve RNA extracted from specific components of fresh-frozen human skin biopsy of 14 subjects. Optimization of the protocol includes a preservation step in RNALater? Solution, the control of specimen temperature, the use of RNase Inhibitors and the time reduction of the staining procedure. The quality of extracted RNA was measured using the percentage of fragments longer than 200 nucleotides (DV200), a more suitable measurement for successful library preparation than the RNA Integrity Number?(RIN). RNA was then enriched using the TruSeq? RNA Access Library Prep Kit (Illumina?) and sequenced on HiSeq??2500 platform (Illumina?). Quality control on RNA sequencing data was adequate to get reliable data for downstream analysis.

The described implemented and optimized protocol can be used for generating transcriptomics data on skin tissues, and it is potentially applicable to other tissues. It can be extended to multicenter studies, due to the introduction of an initial step of preservation of the specimen that allowed the shipment of biological samples.

从人体皮肤活检中获得可靠的组织特异性RNA测序数据代表了研究的重大进展。然而,通过激光捕获显微解剖从新鲜冷冻人体标本中分离特定层的过程的复杂性,皮肤核酸酶的丰富存在和RNA的不稳定性仍然是相关的方法学挑战。我们开发并优化了一种从人体皮肤活检层中提取RNA的方案,并提供了令人满意的质量和数量的mRNA测序数据。该方案包括收集、包埋、冷冻、组织染色和相对优化的步骤,以保存从14名受试者的新鲜冷冻人体皮肤活检的特定成分中提取的RNA。协议的优化包括RNALater?溶液,样品温度的控制,RNase抑制剂的使用和染色程序的时间减少。用长度超过200个核苷酸的片段百分比(DV200)来测量提取的RNA的质量,这比RNA完整性数(RIN)更适合于成功的文库制备。然后用TruSeq?RNA Access Library Prep Kit (Illumina?)并在HiSeq??2500平台(Illumina?)对RNA测序数据的质量控制足以为下游分析提供可靠的数据。所述的实现和优化的方案可用于在皮肤组织上生成转录组学数据,并且它可能适用于其他组织。它可以扩展到多中心研究,因为引入了保存标本的初始步骤,允许运送生物样品。
{"title":"Laser capture microdissection for transcriptomic profiles in human skin biopsies","authors":"Silvia Santoro,&nbsp;Ignazio Diego Lopez,&nbsp;Raffaella Lombardi,&nbsp;Andrea Zauli,&nbsp;Ana Maria Osiceanu,&nbsp;Melissa Sorosina,&nbsp;Ferdinando Clarelli,&nbsp;Silvia Peroni,&nbsp;Daniele Cazzato,&nbsp;Margherita Marchi,&nbsp;Angelo Quattrini,&nbsp;Giancarlo Comi,&nbsp;Raffaele Adolfo Calogero,&nbsp;Giuseppe Lauria,&nbsp;Filippo Martinelli Boneschi","doi":"10.1186/s12867-018-0108-5","DOIUrl":"https://doi.org/10.1186/s12867-018-0108-5","url":null,"abstract":"<p>The acquisition of reliable tissue-specific RNA sequencing data from human skin biopsy represents a major advance in research. However, the complexity of the process of isolation of specific layers from fresh-frozen human specimen by laser capture microdissection, the abundant presence of skin nucleases and RNA instability remain relevant methodological challenges. We developed and optimized a protocol to extract RNA from layers of human skin biopsies and to provide satisfactory quality and amount of mRNA sequencing data.</p><p>The protocol includes steps of collection, embedding, freezing, histological coloration and relative optimization to preserve RNA extracted from specific components of fresh-frozen human skin biopsy of 14 subjects. Optimization of the protocol includes a preservation step in RNALater<sup>?</sup> Solution, the control of specimen temperature, the use of RNase Inhibitors and the time reduction of the staining procedure. The quality of extracted RNA was measured using the percentage of fragments longer than 200 nucleotides (DV<sub>200</sub>), a more suitable measurement for successful library preparation than the RNA Integrity Number?(RIN). RNA was then enriched using the TruSeq<sup>?</sup> RNA Access Library Prep Kit (Illumina<sup>?</sup>) and sequenced on HiSeq<sup>?</sup>?2500 platform (Illumina<sup>?</sup>). Quality control on RNA sequencing data was adequate to get reliable data for downstream analysis.</p><p>The described implemented and optimized protocol can be used for generating transcriptomics data on skin tissues, and it is potentially applicable to other tissues. It can be extended to multicenter studies, due to the introduction of an initial step of preservation of the specimen that allowed the shipment of biological samples.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"19 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2018-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-018-0108-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4755619","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 11
Targeting miR-9 in gastric cancer cells using locked nucleic acid oligonucleotides 利用锁定核酸寡核苷酸靶向胃癌细胞中的miR-9
IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2018-06-07 DOI: 10.1186/s12867-018-0107-6
Joana Filipa Lima, Joana Carvalho, Inês Pinto-Ribeiro, Carina Almeida, Jesper Wengel, Laura Cerqueira, Céu Figueiredo, Carla Oliveira, Nuno Filipe Azevedo

Gastric cancer is the third leading cause of cancer-related mortality worldwide. Recently, it has been demonstrated that gastric cancer cells display a specific miRNA expression profile, with increasing evidence of the role of miRNA-9 in this disease. miRNA-9 upregulation has been shown to influence the expression of E-cadherin-encoding gene, triggering cell motility and invasiveness.

In this study, we designed LNA anti-miRNA oligonucleotides with a complementary sequence to miRNA-9 and tested their properties to both detect and silence the target miRNA. We could identify and visualize the in vitro uptake of low-dosing LNA-based anti-miRNA oligonucleotides without any carrier or transfection agent, as early as 2?h after the addition of the oligonucleotide sequence to the culture medium. Furthermore, we were able to assess the silencing potential of miRNA-9, using different LNA anti-miRNA oligonucleotide designs, and to observe its subsequent effect on E-cadherin expression.

The administration of anti-miRNA sequences even at low-doses, rapidly repressed the target miRNA, and influenced the expression of E-cadherin by significantly increasing its levels.

胃癌是全球癌症相关死亡的第三大原因。最近,研究表明胃癌细胞显示出特定的miRNA表达谱,越来越多的证据表明miRNA-9在该疾病中的作用。miRNA-9上调已被证明会影响e -钙粘蛋白编码基因的表达,引发细胞运动和侵袭性。在这项研究中,我们设计了与miRNA-9序列互补的LNA抗miRNA寡核苷酸,并测试了它们检测和沉默靶miRNA的性能。我们可以识别和可视化低剂量的na基抗mirna寡核苷酸的体外摄取,无需任何载体或转染剂,早在2?H后加入寡核苷酸序列到培养基中。此外,我们能够评估miRNA-9的沉默潜力,使用不同的LNA抗mirna寡核苷酸设计,并观察其对E-cadherin表达的后续影响。即使是低剂量的抗miRNA序列,也能迅速抑制目标miRNA,并通过显著提高E-cadherin的水平来影响其表达。
{"title":"Targeting miR-9 in gastric cancer cells using locked nucleic acid oligonucleotides","authors":"Joana Filipa Lima,&nbsp;Joana Carvalho,&nbsp;Inês Pinto-Ribeiro,&nbsp;Carina Almeida,&nbsp;Jesper Wengel,&nbsp;Laura Cerqueira,&nbsp;Céu Figueiredo,&nbsp;Carla Oliveira,&nbsp;Nuno Filipe Azevedo","doi":"10.1186/s12867-018-0107-6","DOIUrl":"https://doi.org/10.1186/s12867-018-0107-6","url":null,"abstract":"<p>Gastric cancer is the third leading cause of cancer-related mortality worldwide. Recently, it has been demonstrated that gastric cancer cells display a specific miRNA expression profile, with increasing evidence of the role of miRNA-9 in this disease. miRNA-9 upregulation has been shown to influence the expression of E-cadherin-encoding gene, triggering cell motility and invasiveness.</p><p>In this study, we designed LNA anti-miRNA oligonucleotides with a complementary sequence to miRNA-9 and tested their properties to both detect and silence the target miRNA. We could identify and visualize the in vitro uptake of low-dosing LNA-based anti-miRNA oligonucleotides without any carrier or transfection agent, as early as 2?h after the addition of the oligonucleotide sequence to the culture medium. Furthermore, we were able to assess the silencing potential of miRNA-9, using different LNA anti-miRNA oligonucleotide designs, and to observe its subsequent effect on E-cadherin expression.</p><p>The administration of anti-miRNA sequences even at low-doses, rapidly repressed the target miRNA, and influenced the expression of E-cadherin by significantly increasing its levels.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"19 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2018-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-018-0107-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4304653","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 15
Quantitative profiling of BATF family proteins/JUNB/IRF hetero-trimers using Spec-seq 使用Spec-seq定量分析BATF家族蛋白/JUNB/IRF异源三聚体
IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2018-03-27 DOI: 10.1186/s12867-018-0106-7
Yiming K. Chang, Zheng Zuo, Gary D. Stormo

BATF family transcription factors (BATF, BATF2 and BATF3) form hetero-trimers with JUNB and either IRF4 or IRF8 to regulate cell fate in T cells and dendritic cells in vivo. While each combination of the hetero-trimer has a distinct role, some degree of cross-compensation was observed. The basis for the differential actions of IRF4 and IRF8 with BATF factors and JUNB is still unknown. We propose that the differences in function between these hetero-trimers may be caused by differences in their DNA binding preferences. While all three BATF family transcription factors have similar binding preferences when binding as a hetero-dimer with JUNB, the cooperative binding of IRF4 or IRF8 to the hetero-dimer/DNA complex could change the preferences. We used Spec-seq, which allows for the efficient and accurate determination of relative affinity to a large collection of sequences in parallel, to find differences between cooperative DNA binding of IRF4, IRF8 and BATF family members.

We found that without IRF binding, all three hetero-dimer pairs exhibit nearly the same binding preferences to both expected wildtype binding sites TRE (TGA(C/G)TCA) and CRE (TGACGTCA). IRF4 and IRF8 show the very similar DNA binding preferences when binding with any of the three hetero-dimers. No major change of binding preferences was found in the half-sites between different hetero-trimers. IRF proteins bind with substantially lower affinity with either a single nucleotide spacer between IRF and BATF binding site or with an alternative mode of binding in the opposite orientation. In addition, the preference to CRE binding site was reduced with either IRF binding in all BATF–JUNB combinations.

The specificities of BATF, BATF2 and BATF3 are all very similar as are their interactions with IRF4 and IRF8. IRF proteins binding adjacent to BATF sites increases affinity substantially compared to sequences with spacings between the sites, indicating cooperative binding through protein–protein interactions. The preference for the type of BATF binding site, TRE or CRE, is also altered when IRF proteins bind. These in vitro preferences aid in the understanding of in vivo binding activities.

BATF家族转录因子(BATF, BATF2和BATF3)与JUNB和IRF4或IRF8形成异源三聚体,在体内调节T细胞和树突状细胞的细胞命运。虽然异源三聚体的每种组合都有不同的作用,但观察到一定程度的交叉补偿。IRF4和IRF8对BATF因子和JUNB差异作用的基础尚不清楚。我们认为这些异源三聚体的功能差异可能是由它们的DNA结合偏好的差异引起的。虽然所有三种BATF家族转录因子作为异二聚体与JUNB结合时具有相似的结合偏好,但IRF4或IRF8与异二聚体/DNA复合物的协同结合可能改变这种偏好。我们使用了Spec-seq,它允许高效和准确地测定大量序列的相对亲和力,以发现IRF4, IRF8和BATF家族成员的合作DNA结合之间的差异。我们发现,在没有IRF结合的情况下,所有三种异二聚体对预期的野生型结合位点TRE (TGA(C/G)TCA)和CRE (TGACGTCA)都表现出几乎相同的结合偏好。当IRF4和IRF8与三种异二聚体中的任何一种结合时,显示出非常相似的DNA结合偏好。在不同的异源三聚体之间的半位点上没有发现明显的结合偏好变化。IRF蛋白与IRF和BATF结合位点之间的单核苷酸间隔物或与相反方向的另一种结合方式结合的亲和力低得多。此外,在所有BATF-JUNB组合中,IRF结合时对CRE结合位点的偏好降低。BATF、BATF2和BATF3的特异性非常相似,它们与IRF4和IRF8的相互作用也非常相似。与BATF位点相邻结合的IRF蛋白相比,位点之间有间隔的序列亲和力显著增加,表明通过蛋白-蛋白相互作用协同结合。当IRF蛋白结合时,对BATF结合位点(TRE或CRE)类型的偏好也会发生改变。这些体外偏好有助于了解体内结合活性。
{"title":"Quantitative profiling of BATF family proteins/JUNB/IRF hetero-trimers using Spec-seq","authors":"Yiming K. Chang,&nbsp;Zheng Zuo,&nbsp;Gary D. Stormo","doi":"10.1186/s12867-018-0106-7","DOIUrl":"https://doi.org/10.1186/s12867-018-0106-7","url":null,"abstract":"<p>BATF family transcription factors (BATF, BATF2 and BATF3) form hetero-trimers with JUNB and either IRF4 or IRF8 to regulate cell fate in T cells and dendritic cells in vivo. While each combination of the hetero-trimer has a distinct role, some degree of cross-compensation was observed. The basis for the differential actions of IRF4 and IRF8 with BATF factors and JUNB is still unknown. We propose that the differences in function between these hetero-trimers may be caused by differences in their DNA binding preferences. While all three BATF family transcription factors have similar binding preferences when binding as a hetero-dimer with JUNB, the cooperative binding of IRF4 or IRF8 to the hetero-dimer/DNA complex could change the preferences. We used Spec-seq, which allows for the efficient and accurate determination of relative affinity to a large collection of sequences in parallel, to find differences between cooperative DNA binding of IRF4, IRF8 and BATF family members.</p><p>We found that without IRF binding, all three hetero-dimer pairs exhibit nearly the same binding preferences to both expected wildtype binding sites TRE (TGA(C/G)TCA) and CRE (TGACGTCA). IRF4 and IRF8 show the very similar DNA binding preferences when binding with any of the three hetero-dimers. No major change of binding preferences was found in the half-sites between different hetero-trimers. IRF proteins bind with substantially lower affinity with either a single nucleotide spacer between IRF and BATF binding site or with an alternative mode of binding in the opposite orientation. In addition, the preference to CRE binding site was reduced with either IRF binding in all BATF–JUNB combinations.</p><p>The specificities of BATF, BATF2 and BATF3 are all very similar as are their interactions with IRF4 and IRF8. IRF proteins binding adjacent to BATF sites increases affinity substantially compared to sequences with spacings between the sites, indicating cooperative binding through protein–protein interactions. The preference for the type of BATF binding site, TRE or CRE, is also altered when IRF proteins bind. These in vitro preferences aid in the understanding of in vivo binding activities.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"19 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2018-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-018-0106-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5048334","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 10
pH-mediated upregulation of AQP1 gene expression through the Spi-B transcription factor ph通过Spi-B转录因子介导AQP1基因表达上调
IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2018-03-20 DOI: 10.1186/s12867-018-0104-9
Yihui Zhai, Hong Xu, Qian Shen, Franz Schaefer, Claus P. Schmitt, Jing Chen, Haimei Liu, Jialu Liu, Jiaojiao Liu

Bicarbonate-based peritoneal dialysis (PD) fluids enhance the migratory capacity and damage-repair ability of human peritoneal mesothelial cells by upregulating AQP1. However, little is known about the underlying molecular mechanisms.

Here we used HEK-293T cells to investigate the effect of pH on AQP1 gene transcription levels. We found that AQP1 mRNA levels increases with pH. Transfection of HEK-293T cells with luciferase reporter vectors containing different regions of the AQP1 promoter identified an upstream region in the AQP1 gene between ??2200 and –?2300?bp as an enhancer required for pH-mediated regulation of AQP1 expression. Site-directed mutagenesis of this specific promoter region revealed a critical region between ??2257 and ??2251?bp, and gene knock-down experiments and ChIP assays suggested that the Spi-B transcription factor SPIB is involved in pH-mediated regulation of AQP1 expression.

We identified an upstream region in the AQP1 gene and the transcription factor SPIB that are critically involved in pH-mediated regulation of AQP1 expression. These findings provide the basis for further studies on the pH- and buffer-dependent effects of PD fluids on peritoneal membrane integrity and function.

碳酸氢盐基腹膜透析(PD)液通过上调AQP1增强人腹膜间皮细胞的迁移能力和损伤修复能力。然而,人们对其潜在的分子机制知之甚少。我们利用HEK-293T细胞研究pH对AQP1基因转录水平的影响。我们发现AQP1 mRNA水平随ph升高。用含有AQP1启动子不同区域的荧光素酶报告载体转染HEK-293T细胞,发现AQP1基因的上游区域位于??2200 - 2300?bp作为ph介导的AQP1表达调节所需的增强子。对这一特定启动子区域的定点突变揭示了在??2257和2251?基因敲除实验和ChIP分析提示SPIB - b转录因子SPIB参与ph介导的AQP1表达调控。我们确定了AQP1基因的上游区域和转录因子SPIB,它们在ph介导的AQP1表达调控中起关键作用。这些发现为进一步研究PD液的pH和缓冲液对腹膜完整性和功能的影响提供了基础。
{"title":"pH-mediated upregulation of AQP1 gene expression through the Spi-B transcription factor","authors":"Yihui Zhai,&nbsp;Hong Xu,&nbsp;Qian Shen,&nbsp;Franz Schaefer,&nbsp;Claus P. Schmitt,&nbsp;Jing Chen,&nbsp;Haimei Liu,&nbsp;Jialu Liu,&nbsp;Jiaojiao Liu","doi":"10.1186/s12867-018-0104-9","DOIUrl":"https://doi.org/10.1186/s12867-018-0104-9","url":null,"abstract":"<p>Bicarbonate-based peritoneal dialysis (PD) fluids enhance the migratory capacity and damage-repair ability of human peritoneal mesothelial cells by upregulating AQP1. However, little is known about the underlying molecular mechanisms.</p><p>Here we used HEK-293T cells to investigate the effect of pH on <i>AQP1</i> gene transcription levels. We found that <i>AQP1</i> mRNA levels increases with pH. Transfection of HEK-293T cells with luciferase reporter vectors containing different regions of the <i>AQP1</i> promoter identified an upstream region in the <i>AQP1</i> gene between ??2200 and –?2300?bp as an enhancer required for pH-mediated regulation of <i>AQP1</i> expression. Site-directed mutagenesis of this specific promoter region revealed a critical region between ??2257 and ??2251?bp, and gene knock-down experiments and ChIP assays suggested that the Spi-B transcription factor SPIB is involved in pH-mediated regulation of AQP1 expression.</p><p>We identified an upstream region in the <i>AQP1</i> gene and the transcription factor SPIB that are critically involved in pH-mediated regulation of AQP1 expression. These findings provide the basis for further studies on the pH- and buffer-dependent effects of PD fluids on peritoneal membrane integrity and function.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"19 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2018-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-018-0104-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4795248","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone 一种利用pcDNA3主干构建自定义CRISPR Cas9供体载体截断哺乳动物细胞基因的方案
IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2018-03-14 DOI: 10.1186/s12867-018-0105-8
Neftali Vazquez, Lilia Sanchez, Rebecca Marks, Eduardo Martinez, Victor Fanniel, Alma Lopez, Andrea Salinas, Itzel Flores, Jesse Hirschmann, Robert Gilkerson, Erin Schuenzel, Robert Dearth, Reginald Halaby, Wendy Innis-Whitehouse, Megan Keniry

Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone.

We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9?=?CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines.

Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).

集群规则间隔短回文重复(CRISPR) rna引导的适应性免疫系统在原核生物中被发现,以保护细胞免受外来DNA的侵害。CRISPR - Cas9系统已经被修改,并在广泛的生物体中用作基因组编辑工具。在这里,我们提供了一种使用CRISPR Cas9编辑截断哺乳动物细胞中基因的详细方案。我们描述了自定义供体载体建设使用吉布森装配常用的pcDNA3载体为骨干。我们描述了一个循序渐进的方法截断感兴趣的基因在哺乳动物细胞系使用定制供体载体。我们的方法采用2种引导rna,突变Cas9D10A缺口酶(Cas9?=?CRISPR相关序列9),以及用于同源重组的定制供体载体,用可选择的新霉素抗性盒(NPTII)精确截断感兴趣的基因。我们提供了一个详细的方案,如何设计和构建一个定制的供体载体使用吉布森组装(和常用的pcDNA3载体作为主干),允许研究人员获得特定的基因修饰感兴趣(基因截断,基因缺失,表位标记或敲入突变)。用G418 (Geneticin)筛选哺乳动物细胞系中的突变体,并结合几种筛选方法:western blot分析、聚合酶链反应和Sanger测序,从而获得流线型突变体分离。在几种哺乳动物细胞系中进行了原理验证实验。在这里,我们描述了一个详细的方案,利用CRISPR Cas9基因组编辑截断感兴趣的基因,使用常用的表达载体pcDNA3作为供体载体的主干。为定制供体载体设计和构建提供详细的协议将使研究人员能够开发独特的基因组编辑工具。迄今为止,CRISPR Cas9自定义供体载体构建的详细方案有限(Lee等人在Sci Rep:8572, 2015;Ma et al.科学通报,2014(4):44 - 44。定制的供体载体在商业上是可用的,但可能很昂贵。我们的目标是分享这一协议,以帮助研究人员进行遗传研究,这些研究需要定制供体载体用于特定的应用(特定的基因截断,敲入突变和表位标记应用)。
{"title":"A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone","authors":"Neftali Vazquez,&nbsp;Lilia Sanchez,&nbsp;Rebecca Marks,&nbsp;Eduardo Martinez,&nbsp;Victor Fanniel,&nbsp;Alma Lopez,&nbsp;Andrea Salinas,&nbsp;Itzel Flores,&nbsp;Jesse Hirschmann,&nbsp;Robert Gilkerson,&nbsp;Erin Schuenzel,&nbsp;Robert Dearth,&nbsp;Reginald Halaby,&nbsp;Wendy Innis-Whitehouse,&nbsp;Megan Keniry","doi":"10.1186/s12867-018-0105-8","DOIUrl":"https://doi.org/10.1186/s12867-018-0105-8","url":null,"abstract":"<p>Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone.</p><p>We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9?=?CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (<i>NPTII: Neomycin Phosphotransferase II</i>). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain <i>specific</i> gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines.</p><p>Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"19 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2018-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-018-0105-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4582326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 4
Recommendations for mRNA analysis of micro-dissected glomerular tufts from paraffin-embedded human kidney biopsy samples 石蜡包埋人肾活检标本微解剖肾小球簇mRNA分析建议
IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2018-03-13 DOI: 10.1186/s12867-018-0103-x
Clemens L. Bockmeyer, Juliane Wittig, Karen Säuberlich, Philipp Selhausen, Marc Eßer, Philip Zeuschner, Friedrich Modde, Kerstin Amann, Christoph Daniel

Glomeruli are excellent pre-determined natural structures for laser micro-dissection. Compartment-specific glomerular gene expression analysis of formalin-fixed paraffin-embedded renal biopsies could improve research applications. The major challenge for such studies is to obtain good-quality RNA from small amounts of starting material, as applicable for the analysis of glomerular compartments. In this work, we provide data and recommendations for an optimized workflow of glomerular mRNA analysis.

With a proper resolution of the camera and screen provided by the next generation of micro-dissection systems, we are able to separate parietal epithelial cells from glomerular tufts. Selected compartment-specific transcripts (WT1 and GLEPP1 for glomerular tuft as well as PAX2 for parietal epithelial cells) seem to be reliable discriminators for these micro-dissected glomerular substructures. Using the phenol–chloroform extraction and hemalaun-stained sections?(2?μm), high amounts of Bowman’s capsule transections (>?300) reveal sufficient RNA concentrations (>?300?ng mRNA) for further analysis. For comparison, in unstained sections from a number of 60 glomerular transections upwards, a minimum amount of 157?ng mRNA with a reasonable mRNA purity [A260/A280 ratio of 1.5 (1.4/1.7) median (25th/75th percentiles)] was reversely transcribed into cDNA. Comparing the effect of input RNA (20, 60, 150 and 300 micro-dissected glomerular transections), transcript expression of POLR2A significantly correlated when 60 and 150 laser micro-dissected glomerular transections were used for analysis. There was a lower inter-assay coefficient of variability for ADAMTS13, when at least 60 glomerular transections were used. According to the algorithms of geNormPlus and NormFinder, PGK1 and PPIA are more stable glomerular reference transcripts compared to GUSB, GAPDH, POLR2A, RPLPO, TBP, B2M, ACTB, 18SrRNA and HMBS.

Our approach implements compartment-specific glomerular mRNA expression analysis into research applications, even regarding glomerular substructures like parietal epithelial cells. We recommend using of at least 60 micro-dissected unstained glomerular or 300 hemalaun-stained Bowman’s capsule transections to obtain sufficient input mRNA for reproducible results. Hereby, the range of RNA concentrations in 60 micro-dissected glomeruli is low and appropriate normalization of Cq values using our suggested reference transcripts (PGK1 and PPIA) allows compensation with respect to different amounts of RNA purity and quantity.

肾小球是用于激光显微解剖的优良的预先确定的自然结构。福尔马林固定石蜡包埋肾活检细胞间室特异性肾小球基因表达分析可提高研究应用。这类研究面临的主要挑战是如何从少量起始材料中获得高质量的RNA,以用于肾小球室的分析。在这项工作中,我们为优化肾小球mRNA分析工作流程提供了数据和建议。利用下一代显微解剖系统提供的适当分辨率的相机和屏幕,我们能够从肾小球簇中分离壁上皮细胞。选定的室特异性转录物(肾小球簇的WT1和GLEPP1以及壁上皮细胞的PAX2)似乎是这些微解剖肾小球亚结构的可靠鉴别物。使用苯酚-氯仿提取和血细胞染色切片(2 μm),大量的鲍曼囊切片(>?300)显示出足够的RNA浓度(>?300?ng mRNA)作进一步分析。相比之下,在60例以上的未染色肾小球切片中,至少有157?以合理的mRNA纯度[A260/A280比值为1.5(1.4/1.7)中位数(25 /75百分位数)]逆转录到cDNA中。对比输入RNA(20、60、150和300个微解剖肾小球切片)的影响,用60和150个激光微解剖肾小球切片进行分析时,POLR2A转录物表达显著相关。当使用至少60个肾小球横断时,ADAMTS13的测定间变异性系数较低。根据geNormPlus和NormFinder算法,与GUSB、GAPDH、POLR2A、RPLPO、TBP、B2M、ACTB、18SrRNA和HMBS相比,PGK1和PPIA是更稳定的肾小球参考转录物。我们的方法将室特异性肾小球mRNA表达分析应用于研究,甚至涉及肾小球亚结构,如壁上皮细胞。我们建议使用至少60个未染色的微解剖肾小球或300个未染色的鲍曼囊切片来获得足够的输入mRNA,以获得可重复的结果。因此,60个微解剖肾小球的RNA浓度范围很低,使用我们建议的参考转录物(PGK1和PPIA)适当正常化Cq值可以补偿不同数量的RNA纯度和数量。
{"title":"Recommendations for mRNA analysis of micro-dissected glomerular tufts from paraffin-embedded human kidney biopsy samples","authors":"Clemens L. Bockmeyer,&nbsp;Juliane Wittig,&nbsp;Karen Säuberlich,&nbsp;Philipp Selhausen,&nbsp;Marc Eßer,&nbsp;Philip Zeuschner,&nbsp;Friedrich Modde,&nbsp;Kerstin Amann,&nbsp;Christoph Daniel","doi":"10.1186/s12867-018-0103-x","DOIUrl":"https://doi.org/10.1186/s12867-018-0103-x","url":null,"abstract":"<p>Glomeruli are excellent pre-determined natural structures for laser micro-dissection. Compartment-specific glomerular gene expression analysis of formalin-fixed paraffin-embedded renal biopsies could improve research applications. The major challenge for such studies is to obtain good-quality RNA from small amounts of starting material, as applicable for the analysis of glomerular compartments. In this work, we provide data and recommendations for an optimized workflow of glomerular mRNA analysis.</p><p>With a proper resolution of the camera and screen provided by the next generation of micro-dissection systems, we are able to separate parietal epithelial cells from glomerular tufts. Selected compartment-specific transcripts (WT1 and GLEPP1 for glomerular tuft as well as PAX2 for parietal epithelial cells) seem to be reliable discriminators for these micro-dissected glomerular substructures. Using the phenol–chloroform extraction and hemalaun-stained sections?(2?μm), high amounts of Bowman’s capsule transections (&gt;?300) reveal sufficient RNA concentrations (&gt;?300?ng mRNA) for further analysis. For comparison, in unstained sections from a number of 60 glomerular transections upwards, a minimum amount of 157?ng mRNA with a reasonable mRNA purity [A260/A280 ratio of 1.5 (1.4/1.7) median (25th/75th percentiles)] was reversely transcribed into cDNA. Comparing the effect of input RNA (20, 60, 150 and 300 micro-dissected glomerular transections), transcript expression of POLR2A significantly correlated when 60 and 150 laser micro-dissected glomerular transections were used for analysis. There was a lower inter-assay coefficient of variability for ADAMTS13, when at least 60 glomerular transections were used. According to the algorithms of geNormPlus and NormFinder, PGK1 and PPIA are more stable glomerular reference transcripts compared to GUSB, GAPDH, POLR2A, RPLPO, TBP, B2M, ACTB, 18SrRNA and HMBS.</p><p>Our approach implements compartment-specific glomerular mRNA expression analysis into research applications, even regarding glomerular substructures like parietal epithelial cells. We recommend using of at least 60 micro-dissected unstained glomerular or 300 hemalaun-stained Bowman’s capsule transections to obtain sufficient input mRNA for reproducible results. Hereby, the range of RNA concentrations in 60 micro-dissected glomeruli is low and appropriate normalization of C<sub>q</sub> values using our suggested reference transcripts (PGK1 and PPIA) allows compensation with respect to different amounts of RNA purity and quantity.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"19 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2018-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-018-0103-x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4548146","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 6
Nutrient depletion and TOR inhibition induce 18S and 25S ribosomal RNAs resistant to a 5′-phosphate-dependent exonuclease in Candida albicans and other yeasts 在白色念珠菌和其他酵母菌中,营养消耗和TOR抑制诱导18S和25S核糖体rna抵抗5 ' -磷酸依赖性核酸外切酶
IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2018-01-19 DOI: 10.1186/s12867-018-0102-y
Jacob Fleischmann, Miguel A. Rocha

Messenger RNA (mRNA) represents a small percentage of RNAs in a cell, with ribosomal RNA (rRNA) making up the bulk of it. To isolate mRNA from eukaryotes, typically poly-A selection is carried out. Recently, a 5′-phosphate-dependent, 5′→3′ processive exonuclease called Terminator has become available. It will digest only RNA that has a 5′-monophosphate end and therefore it is very useful to eliminate most of rRNAs in cell.

We have found that in the pathogenic yeast Candida albicans, while 18S and 25S components isolated from yeast in robust growth phase are easily eliminated by Terminator, those isolated from cells in the nutritionally diminished stationary phase, become resistant to digestion by this enzyme. Additional digestions with alkaline phosphatase, tobacco pyrophosphatase combined with Terminator point toward the 5′-prime end of 18S and 25S as the source of this resistance. Inhibition of TOR by rapamycin also induces resistance by these molecules. We also find that these molecules are incorporated into the ribosome and are not just produced incidentally. Finally, we show that three other yeasts show the same behavior.

Digestion of RNA by Terminator has revealed 18S and 25S rRNA molecules different from the accepted processed ones seen in ribosome generation. The reason for these molecules and the underlying mechanism for their formation is unknown. The preservation of this behavior across these yeasts suggests a useful biological role for it, worthy of further inquiry.

信使RNA (mRNA)代表了细胞中一小部分RNA,核糖体RNA (rRNA)构成了大部分RNA。为了从真核生物中分离mRNA,通常进行poly-A选择。最近,一种5 ' -磷酸依赖的5 '→3 '过程外切酶被称为终结者。它只会消化具有5 ' -单磷酸末端的RNA,因此它对消除细胞中的大多数rrna非常有用。我们发现,在白色念珠菌中,从健壮生长期的酵母中分离出的18S和25S组分很容易被终结者酶消除,而从营养减少的静止期细胞中分离出的18S和25S组分对该酶产生抗性。碱性磷酸酶、烟草焦磷酸酶联合终结者酶的额外消化指向18S和25S的5 ' - '端作为这种抗性的来源。雷帕霉素对TOR的抑制也会诱导这些分子产生耐药性。我们还发现这些分子与核糖体结合,并不是偶然产生的。最后,我们证明了另外三种酵母也表现出同样的行为。用终结者酶切RNA,发现18S和25S rRNA分子不同于核糖体生成中被接受的加工过的RNA分子。这些分子产生的原因及其形成的潜在机制尚不清楚。在这些酵母中保存这种行为表明它具有有用的生物学作用,值得进一步研究。
{"title":"Nutrient depletion and TOR inhibition induce 18S and 25S ribosomal RNAs resistant to a 5′-phosphate-dependent exonuclease in Candida albicans and other yeasts","authors":"Jacob Fleischmann,&nbsp;Miguel A. Rocha","doi":"10.1186/s12867-018-0102-y","DOIUrl":"https://doi.org/10.1186/s12867-018-0102-y","url":null,"abstract":"<p>Messenger RNA (mRNA) represents a small percentage of RNAs in a cell, with ribosomal RNA (rRNA) making up the bulk of it. To isolate mRNA from eukaryotes, typically poly-A selection is carried out. Recently, a 5′-phosphate-dependent, 5′→3′ processive exonuclease called Terminator has become available. It will digest only RNA that has a 5′-monophosphate end and therefore it is very useful to eliminate most of rRNAs in cell.</p><p>We have found that in the pathogenic yeast <i>Candida albicans</i>, while 18S and 25S components isolated from yeast in robust growth phase are easily eliminated by Terminator, those isolated from cells in the nutritionally diminished stationary phase, become resistant to digestion by this enzyme. Additional digestions with alkaline phosphatase, tobacco pyrophosphatase combined with Terminator point toward the 5′-prime end of 18S and 25S as the source of this resistance. Inhibition of TOR by rapamycin also induces resistance by these molecules. We also find that these molecules are incorporated into the ribosome and are not just produced incidentally. Finally, we show that three other yeasts show the same behavior.</p><p>Digestion of RNA by Terminator has revealed 18S and 25S rRNA molecules different from the accepted processed ones seen in ribosome generation. The reason for these molecules and the underlying mechanism for their formation is unknown. The preservation of this behavior across these yeasts suggests a useful biological role for it, worthy of further inquiry.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"19 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2018-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-018-0102-y","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5048139","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 7
An optimized rapid bisulfite conversion method with high recovery of cell-free DNA 一种优化的亚硫酸氢盐快速转化无细胞DNA的高回收率方法
IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2017-12-19 DOI: 10.1186/s12867-017-0101-4
Shaohua Yi, Fei Long, Juanbo Cheng, Daixin Huang

Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA.

We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30?min at 70?°C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods.

The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.

游离DNA的甲基化分析是肿瘤诊断、监测和预后的一个令人鼓舞的工具。由于血浆中游离DNA的含量极低,甲基化分析的敏感性是一个非常重要的问题。目前大多数DNA甲基化分析方法都是基于亚硫酸亚硫酸盐介导的胞嘧啶脱氨作用在胞嘧啶和5-甲基胞嘧啶中的差异。然而,基于现有方法的亚硫酸盐转化DNA的恢复对于无细胞DNA的甲基化分析非常差。我们优化了亚硫酸氢盐转化关键步骤的快速方法,并获得了高的无细胞DNA回收率。采用快速脱氨和碱性脱硫相结合的方法,在硅胶柱上提纯DNA。采用微滴数字PCR技术对亚硫酸氢盐处理DNA的转化效率和回收率进行了研究。优化后的反应在30分钟内完成了胞嘧啶的完全转化。最低70?°C,亚硫酸处理的无细胞DNA的回收率约为65%,高于目前的方法。该方法允许从低水平亚硫酸处理的游离DNA中获得高回收率,提高了从游离DNA中进行甲基化检测的分析灵敏度。
{"title":"An optimized rapid bisulfite conversion method with high recovery of cell-free DNA","authors":"Shaohua Yi,&nbsp;Fei Long,&nbsp;Juanbo Cheng,&nbsp;Daixin Huang","doi":"10.1186/s12867-017-0101-4","DOIUrl":"https://doi.org/10.1186/s12867-017-0101-4","url":null,"abstract":"<p>Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA.</p><p>We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30?min at 70?°C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods.</p><p>The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.</p>","PeriodicalId":497,"journal":{"name":"BMC Molecular Biology","volume":"18 1","pages":""},"PeriodicalIF":2.946,"publicationDate":"2017-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s12867-017-0101-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4747388","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 14
期刊
BMC Molecular Biology
全部 Acc. Chem. Res. ACS Applied Bio Materials ACS Appl. Electron. Mater. ACS Appl. Energy Mater. ACS Appl. Mater. Interfaces ACS Appl. Nano Mater. ACS Appl. Polym. Mater. ACS BIOMATER-SCI ENG ACS Catal. ACS Cent. Sci. ACS Chem. Biol. ACS Chemical Health & Safety ACS Chem. Neurosci. ACS Comb. Sci. ACS Earth Space Chem. ACS Energy Lett. ACS Infect. Dis. ACS Macro Lett. ACS Mater. Lett. ACS Med. Chem. Lett. ACS Nano ACS Omega ACS Photonics ACS Sens. ACS Sustainable Chem. Eng. ACS Synth. Biol. Anal. Chem. BIOCHEMISTRY-US Bioconjugate Chem. BIOMACROMOLECULES Chem. Res. Toxicol. Chem. Rev. Chem. Mater. CRYST GROWTH DES ENERG FUEL Environ. Sci. Technol. Environ. Sci. Technol. Lett. Eur. J. Inorg. Chem. IND ENG CHEM RES Inorg. Chem. J. Agric. Food. Chem. J. Chem. Eng. Data J. Chem. Educ. J. Chem. Inf. Model. J. Chem. Theory Comput. J. Med. Chem. J. Nat. Prod. J PROTEOME RES J. Am. Chem. Soc. LANGMUIR MACROMOLECULES Mol. Pharmaceutics Nano Lett. Org. Lett. ORG PROCESS RES DEV ORGANOMETALLICS J. Org. Chem. J. Phys. Chem. J. Phys. Chem. A J. Phys. Chem. B J. Phys. Chem. C J. Phys. Chem. Lett. Analyst Anal. Methods Biomater. Sci. Catal. Sci. Technol. Chem. Commun. Chem. Soc. Rev. CHEM EDUC RES PRACT CRYSTENGCOMM Dalton Trans. Energy Environ. Sci. ENVIRON SCI-NANO ENVIRON SCI-PROC IMP ENVIRON SCI-WAT RES Faraday Discuss. Food Funct. Green Chem. Inorg. Chem. Front. Integr. Biol. J. Anal. At. Spectrom. J. Mater. Chem. A J. Mater. Chem. B J. Mater. Chem. C Lab Chip Mater. Chem. Front. Mater. Horiz. MEDCHEMCOMM Metallomics Mol. Biosyst. Mol. Syst. Des. Eng. Nanoscale Nanoscale Horiz. Nat. Prod. Rep. New J. Chem. Org. Biomol. Chem. Org. Chem. Front. PHOTOCH PHOTOBIO SCI PCCP Polym. Chem.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1