Identification of differentially expressed genes by mutually subtracted RNA fingerprinting

A mutually subtracted RNA fingerprinting (SuRF) method has been developed that allows efficient identification of differentially expressed sequence tags between two samples. Mutual subtractions of two RNA samples are achieved by first synthesizing cDNAs using oligo(dT) coupled with magnetic beads which are then reciprocally hybridized to starting RNA samples to remove common mRNAs between them. The second step involves differential fingerprinting of the subtracted RNA samples by polymerase chain reaction with specially designed degenerate primers. SuRF was applied to identify alteration in gene expression pertinent to osteogenic sarcoma which was achieved by employing the method between FOB (an immortalized fetal osteoblast) and MG63 (an osteosarcoma) cell lines. An estimated 10% of the total expressed genes in these two cell types were screened by the method. This analysis identified 96 differentially expressed sequences, none of which was identified repeatedly. A subset of these sequences was subsequently confirmed to have differential expression between the two cell types. Removal of common mRNAs prior to differential display should diminish redundant identification of abundant genes and increase the chance of identifying rare differentially expressed genes.     

Identification of hypoxia-responsive messengers expressed in human microvascular endothelial cells using differential display RT-PCR.

In order to identify new genes overexpressed in endothelial cells exposed to hypoxia, differential display RT-PCR was performed on total RNA extracted from human microvascular endothelial cells incubated under hypoxia or under normoxic conditions. Northern blot and reverse Northern blot analyses were used to confirm the results. Sequences corresponding to tissue inhibitor of metalloproteinase-1, prostate tumor inducing factor-1, enolase-alpha and prothymosin-alpha were evidenced as overexpressed in hypoxia. These results were confirmed by Western blot and immunofluorescence experiments. Moreover, several elements homologous to partial sequences of cDNA (expressed sequence tag) were also identified, as well as unknown cDNA sequences. The present study suggests that hypoxia can change the expression of numerous genes in endothelial cells, and that mRNA differential display is useful for cloning known and unknown hypoxia-responsive genes.     

Differential gene expression during Trypanosoma cruzi metacyclogenesis

The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE). The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes) resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells), while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.     

高温诱导金黄仓鼠胚神经上皮细胞基因的差异表达

以mRNA差异显示法研究高温作用后金黄仓鼠胚神经上皮细胞基因的差异表达。结果表明,高温处理后第16h,从鼠胚神经上皮细胞基因表达中检出两条良好重复性差异序列。该差异序列为未知序列,属新EST,提示高温可能诱导金黄仓鼠胚神经上皮细胞基因表达发生变化。     

中外两品种鸡胸肌组织差异表达基因的研究

运用mRNA差异显示技术对北京油鸡和AA肉鸡胸肌组织基因的差异表达进行研究, 从分子水平分析导致两品种肌肉组织基因差异表达的机制。通过反向Northern dot blot技术验证共筛选出差异表达基因7条, 经与GenBank数据库进行相似性比对, S1与HMGN3基因有较高同源性; S3与ChEST294a8有很高同源性, 但功能未知; S4和S5与鸡的磷酸葡萄糖变位酶Ⅰ同源性很高; S6和S2与已有核酸数据库中的基因克隆或EST具有较高同源性, 为已知的EST, 但功能未知; 序列S7未在数据中发现同源序列, 可以确定其在AA肉鸡中特异表达,确定为新发现的EST(GenBank登录号: EU594549)。为进一步研究中外两品种鸡胸肌组织基因差异表达机制奠定了基础。     

Altered levels of histone deacetylase OsHDT1 affect differential gene expression patterns in hybrid rice

Hybrids between different inbred varieties display novel patterns of gene expression resulted from parental variation in allelic nucleotide sequences. To study the function of chromatin regulators in hybrid gene expression, the histone deacetylase gene OsHDT1 whose expression displayed a circadian rhythm was over-expressed or inactivated by RNAi in an elite rice parent. Increased OsHDT1 expression did not affect plant growth in the parent but led to early flowering in the hybrid. Nonadditive up-regulation of key flowering time genes was found to be related to flowering time of the hybrid. Over-expression of OsHDT1 repressed the nonadditive expression of the key flowering repressors in the hybrid (i.e. OsGI and Hd1) inducing early flowering. Analysis of histone acetylation suggested that OsHDT1 over-expression might promote deacetylation on OsGI and Hd1 chromatin during the peak expression phase. High throughput differential gene expression analysis revealed that altered OsHDT1 levels affected nonadditive expression of many genes in the hybrid. These data demonstrate that nonadditive gene expression was involved in flowering time control in the hybrid rice and that OsHDT1 level was important for nonadditive or differential expression of many genes including the flowering time genes, suggesting that OsHDT1 may be involved in epigenetic control of parental genome interaction for differential gene expression.     

Cataloging altered gene expression in young and senescent cells using enhanced differential display.

Recently, a novel PCR-based technique, differential display (DD), has facilitated the study of differentially expressed genes at the mRNA level. We report here an improved version of DD, which we call Enhanced Differential Display (EDD). We have modified the technique to enhance reproducibility and to facilitate sequencing and cloning. Using EDD, we have generated and verified a catalog of genes that are differentially expressed between young and senescent human diploid fibroblasts (HDF). From 168 genetags that were identified initially, 84 could be sequenced directly from PCR amplified bands. These sequences represent 27 known genes and 37 novel genes. By Northern blot analysis we have confirmed the differential expression of a total of 23 genes (12 known, 11 novel), while 19 (seven known, 12 novel) did not show differential expression. Several of the known genes were previously observed by others to be differentially expressed between young and senescent fibroblasts, thereby validating the technique.     

Changes in mRNA expression in mouse postnatal cochlea by differential display method.

We compared mRNA expression by mRNA differential display method in postnatal day 11 (P11), P13 and adult C3H/HeJ mouse cochlea. Forty-seven bands were differentially displayed on polyacrylamide gel when 27 patterns of PCR primer sets were used, and 24 of them showed a remarkable increase within only two days (P11 and P13). DNA sequences of the bands were analyzed for homology to known genes using the BLAST search. Most of the clones were identical to sequences of functions unknown. However, one of the clones showing an increase of mRNA expression between P11 and P13 was identified as mouse TIS7 which is known to markedly increase during differentiation of PC-12 cells to neurons by NGF-treatment. TIS7 expression may be involved in differentiation of neuronal progenitor cells to spiral ganglion cells by the initial sound input. The comparison among P11, P13 and adult mouse cochlear mRNA expressions investigated the genes involved in the various growing stages of the postnatal cochlea.     

用mRNA差异显示法分离冷胁迫香蕉幼苗叶片内水杨酸诱导表达的基因

用mRNA差异显示法对7℃低温胁迫3 d后的香蕉幼苗叶片进行研究,回收了18条在低温下水杨酸(salicylic acid,SA)诱导的差异带;反向Northern杂交证明,SA在低温胁迫下能诱导7条差异带高表达.对其中最显著表达的两条差异带(G和A)进行克隆和测序,结果显示,G片段序列与大豆在冷胁迫环境下两个高表达基因片段的部分序列有92%同源性;A片段未发现其同源性基因片段.     

Differential display: Identifying genes involved in cardiomyocyte proliferation

An estimated 15,000 different mRNA species are expressed in a typical mammalian cell. The differential expression of mRNAs in both a temporal and cell-specific manner determines the fate of the cell and creates the organism. Analysis of this differential gene expression has become a central aim of many laboratories attempting to understand the mechanisms underlying various biological processes. Currently, we are using a technique called differential display to analyze the differential expression of genes in cardiomyocytes. Differential display is a rapid and powerful technique that was introduced by Liang and Pardee in 1992. Since that time, it has been successfully applied by several groups, and it is quickly becoming a standard method for studying differential gene expression. Here, we present a detailed article discussing the differential display methodology and how we have utilized it to identify potential genes involved in cardiomyocyte proliferation. Furthermore, we have provided a list of materials and supplied examples of data obtained, in an effort to allow the reader to perform the technique with success in their own laboratory.     

草甘膦诱导表达的大豆与棉花EST序列的分离

利用差异显示PCR方法分离了63个草甘膦诱导后在大豆和棉花中差异表达的片段,测序分析结果表明属于33个草甘膦诱导的大豆和棉花EST序列。通过在GenBank中进一步比时研究发现:约85％的EST序列与水杨酸、冷、创伤、氧化等非生物胁迫诱导后表达库中的EST序列有高达95％以上的同源性,由此可推测这些基因参与了植物对非生物胁迫的反应过程。草甘膦诱导后高表达EST、序列的获得将有利于进一步分离相关非生物胁迫诱导表达基因及启动子,研究其转录调控的机理,有望建立草甘膦诱导系统。从而解决组成型表达造成外源基因在植物体所有发育阶段和所有组织部位表达,造成植物体能量浪费。     

Enhanced expression of the LDH-A gene after gravity-changing stress in human RSa cells.

A major issue in radiation and space biology is whether gene expression levels are altered in cells exposed to gravity-changing stress. In the present study, genes up- or down-regulated in radiation-sensitive human RSa cells cultured under gravity-changing conditions, were identified using a PCR-based mRNA differential display method. Exposure of cells to gravity-changing stress was performed by free-fall with a drop-shaft facility or by an airplane-conducted parabolic flight. Among the candidates for gravity-changing stress-responsive genes obtained by the differential display analysis, the lactate dehydrogenase A gene (LDH-A) was confirmed by Northern blotting analysis to exhibit increased expression levels. The gravity-changing stress consisted of a combination of microgravity and hypergravity. However, exposure of the cells to hypergravity produced by centrifuge only slightly affected the LDH-A mRNA expression. Thus, LDH-A was found to be a candidate for the genes which play a role in the cellular response to gravity-changing stress, and mainly to microgravity.     

北京油鸡与AA肉鸡脂肪组织差异表达基因的研究

运用mRNA差异显示技术对AA肉鸡和北京油鸡脂肪组织基因的差异表达进行研究,从分子遗传学角度分析导致两品种脂肪组织差异表达的原因,对了解性状形成的遗传基础和调控机理是十分必要的。通过反Northern杂交验证共筛选出脂肪组织差异表达基因10条,经与GenBank数据库进行相似性比对,XF1 与已知基因有较高同源性,该基因是人类cDNA全长开放阅读框（ORF）的一段; XF2、YF1、YF2及YF4经与nr数据库进行同源性比对,均可找到同源性较高的基因,但功能未知;XF4 与克隆人类胎盘CL0BA010ZF08基因的一段cDNA序列同源性为83%;YF3与预测原鸡MLL5 (LOC417712)基因有一定的同源性,目前尚无功能报道;XF5和YF5 与原鸡高迁移率族蛋白（HMGN3）有较高同源性;XF3 在nr库中未找到同源序列,确定为新发现的EST,提交数据库获得GenBank登录号(Accession number: EU594549)。为进一步研究北京油鸡与AA肉鸡脂肪组织差异基因的功能与脂肪发育的关系奠定基础。     

小麦光温敏核雄性不育相关基因的G-box家族引物差式分析

用G-box家族引物对小麦光温敏核雄性不育系农大3338在可育与不育光温条件下进行mRNA差异显示,结果表明,在育性转换时期,这两种条件下的基因表达存在显著差异。回收了12个质的差异片段并进行反Northern印迹杂交验证,然后对5个阳性克隆片段HT1-G10、HT1-G3、HT2-G2、HT1-G4和HT2-G5进行了测序,同源比较显示：HT1-G10与小麦（Triticum aestivum）叶绿体基因rbcL和atpB的部分序列高度同源（96％）;HT1-G3与小麦（Triticum aestivum）组蛋白H2A基因高度同源（88％）;另3个片段为新基因片段。对这些基因片段的分析为揭示光温敏核雄性不育的发育机理提供了一些有效证据。     

Differential display cloning of genes induced in regenerating neurons

Mammalian adult motor and sensory neurons are thought to reexpress a complement of genes that are originally expressed during development when they regenerate following injury. Therefore, one strategy for identifying key genes involved in development of the peripheral nervous system is to identify those genes reexpressed in the regenerating system. To test this hypothesis, we used the single-base anchor method of mRNA differential display to study changes in gene expression in regenerating adult mammalian sensory neurons. From an initial sample of 36 different primer combinations [3 oligo(dT)M primers x 12 arbitrary 13-mers], 6 candidate upregulated and 6 candidate downregulated genes were identified. Candidate genes were screened by the reverse Northern blot method to eliminate obvious false positives and the three remaining candidates cloned and sequenced. In addition to comparing isolated sequences with the public databases, sequences were also compared with assembled clusters of expressed sequence tag sequences, enabling extension of the sequence data by more than a kilobase from the isolated 3' cDNA fragments. Ultimate confirmation of differential expression was carried out by in situ hybridization using 45-base oligonucleotides complementary to the predicted 5'-3' orientation of the corresponding mRNAs of all three cDNAs. Two, LA12.2 and LC12, were definitively confirmed as induced in regenerating neurons. The sequence of LC12 is identical to that of the secreted protein Reg-2 and a detailed study of the functions of this secreted protein in neural development and regeneration has been published (F. J. Livesey, J. A. O'Brien, M. Li, A. G. Smith, L. J. Murphy, and S. P. Hunt, 1997, Nature 390, 614-618). The LA12.2 gene is currently being characterized, the available sequence of this cDNA being novel.     

基因网络中基因关系图谱的构建及其应用

尽快破解基因组所包含基因的功能是一项费力但又很重要的工作。一个基因功能的实现依赖于该基因与其它基因间的相互作用。基因网络是一组基因的集合体,这些基因通过相互协作来控制生物体重要的生命过程。通过基因敲除、RNA干扰或其它方法改变基因网络中某个基因的表达水平,将会引起该网络中其它基因表达水平的变化。而这种变化可以方便地通过基因表达差异显示技术检测相应mRNA含量变化来反映。因此,将这两类方法组合在一起,可以在基因组水平上有效地检测出基因网络中的基因关系。这种策略对基因功能研究方法是一个重要补充。