Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes

Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.     

雌核发育银鲫原肠期胚胎和尾芽期胚胎差异表达基因的呈现

构建了雌核发育银鲫原肠期胚胎和尾芽期胚胎间的抑制性差减杂交cDNA质粒文库。对原肠期’739个和尾芽期816个PCR阳性克隆进行斑点杂交,得到72个原肠期和98个尾芽期斑点杂交阳性克隆。测序和基因数据库比对结果表明：72个原肠期斑点杂交阳性克隆中,包括19个已知基因的cDNA片段和31个没有同源性的cDNA片段;98个尾芽期斑点杂交阳性克隆中,包括52个已知基因的cDNA片段和37个没有同源性的cDNA片段。采用虚拟Northern杂交和RT-PCR证实了部分基因在银鲫胚胎发育过程中的差异表达。这些差异表达基因的呈现为进一步研究银鲫胚胎发育的分子机制奠定了基础。     

Functional genomics of maize submergence tolerance and cloning of the related gene Sicyp51

In the middle and lower Yangtze River area, the major corn-growing region of South China, seasonal rainfall greatly affects maize plantation. Maize seed-lings meet with excessive precipitation and low tem-perature in spring, and when they grow up to begin flowering, they usually encounter Mei-yu storm ac-companied by hot days. At the same time, bad irriga-tion system and a higher level of underground water cause waterlogging, which further leads to yield losses. In order to reveal the molecu…     

Functional genomics of maize submergence tolerance and cloning of the related gene <Emphasis Type="Italic">Sicyp51</Emphasis>

In this study, SSH (Suppression Subtractive Hybridization) and cDNA microarray were used to identify genes associated with waterlogging response of maize roots. Mo17 and Hz32 are two maize inbred lines with differential tolerance to hypoxia. Seedlings of the inbred lines with two leaves were submerged in hypoxia buffer. SSH libraries were constructed with cDNA samples from roots. Both forward and reverse subtractions were performed for each inbred line, and 105 positive clones induced by hypoxia were selected by differential screening. The treated and control message RNA were hybridized with the cDNA microarray of Mo17, sequentially, 57 of 3-fold differentially expressed clones were obtained. A total of 162 positive clones were all sequenced. Bioinformatics analysis showed these positive clones represent 85 TUGs, including genes involved in several biochemistry pathways, such as glycolysis, protection, signal transduction, cell construction and energy metabolism and 41 EST with unknown function. Comparison between Mo17 and Hz32 indicates that genes related to hypoxia tolerance have different expression patterns in submerged roots. Several positive clones' expression patterns were revealed by Northern or RT-PCR, and a new gene (Sicyp51), which may contribute to hypoxia tolerance, was identified.     

The T cell receptor beta chain genes are located on chromosome 6 in mice and chromosome 7 in humans

Homologous clones that encode the beta chain of the T cell antigen receptor have been isolated recently from both murine and human cDNA libraries. These cDNA clones have been used in connection with interspecies hybrid cell lines to determine that the murine T cell receptor gene is located on chromosome 6 and the human gene on chromosome 7. In situ hybridization confirms these data and further localizes these genes to band B of chromosome 6 in the mouse and bands 7p13-21 in the human genome. The organization of the T cell antigen receptor J beta gene segments and C beta genes appears to be conserved, since very few intraspecies polymorphisms of restriction fragment length have been detected in either mouse or human DNA.     

血管紧张素Ⅱ诱导心肌成纤维细胞表达上调基因的分离和鉴定

为了对成年大鼠心肌成纤维细胞（cardiac fibroblasts,CF）受血管紧张素Ⅱ（angiotensin Ⅱ,AngⅡ）刺激后上调基因表达谱进行筛选及分析,以受AngⅡ刺激CF为实验方,未刺激CF为驱动方,进行抑制消减杂交（suppression subtractive hybridization,SSH）,建立消减cDNA文库。经斑点杂交筛选文库后将表达变化显著的部分阳性克隆测序及同源性分析,共获得19个上调表达的基因,分别与细胞外基质、细胞周期、胞内信号转导、细胞骨架及细胞代谢等功能相关,并克隆到7个新的基因表达序列标签（expressed sequence tags,EST）。我们的数据证实了SSH可以有效地克隆成年大鼠CF受AngⅡ刺激后上调表达基因,对这些基因的研究将有助于阐明心肌重塑的分子机制。     

Identification and characterization of differential gene expression in the mesocarp and kernel of oil palm nuts using suppression subtractive hybridization

Suppression subtracted hybridization (SSH) and dot blotting were used to identify differential gene expression in the mesocarp and kernel of oil palm nuts. The different types of nut tissue show differences in fatty acid anabolism and the synthesis of other important compounds. In total, 302 clones from forward SSH libraries and 238 clones from reverse SSH libraries were identified following differential screening, respectively. Among these, 120 clones from the forward SSH library and 81 clones from the reverse SSH library, showed tenfold or more differential expression levels, and were sequenced. Sequence analysis revealed that 76 clones (28 from the forward SSH library and 48 from the reverse SSH library) represent non-redundant cDNA inserts. The differential expression of 39 subset genes in the two different tissues was further confirmed by RT-PCR analysis. Functionally annotated blasting against the GenBank non-redundant protein database classified all 76 candidate genes into six categories, according to their putative functions. Interestingly, our results show that a group of significantly differentially expressed genes are involved in processes associated with oil palm nut maturation, such as the synthesis of medium-chain saturated fatty acids and phytic acid, nut development, and stress/defense responses. This study describes some relationships between gene expression and metabolic pathways in mature oil palm nuts, and contributes to our understanding of oil palm nut ESTs.     

Suppression subtractive hybridization identifies differentially expressed genes in Brassica napus chlorophyll-reduced mutant

Suppressive subtraction hybridization (SSH) was used to identify differentially expressed genes caused by a chlorophyll-reduced mutation in B. napus. The cDNA fragments, derived from SSH positive subtractive library (tester: normal wild type, driver: mutant) were cloned into pMD18-T vector. Two hundred SSH cDNA clones were screened by dot blot array, and 151 clones were identified as differentially expressed cDNA fragments in Cr3529 line. Thirty-six positive clones which showed marked expression differences were selected and sequenced. After redundant cDNAs were removed, 33 differentially expressed unique cDNA section clones were obtained. Among the 33 clones, two clones possess different parts of the cDNA sequence of the same gene coding geranylgeranyl reductase, four clones belong to unknown proteins, and the rest share homology to genes of diverse class. Sequence analysis showed that at least 12 genes were discovered to be related to the photosynthesis, seven of them coded the proteins which belong to the subunit of photosystem 2. RNA gel blot analysis showed that compared with 3529, the gene expression of the chlorophyll a/b-binding protein Lhcb2 in photosystem 2 declined markedly in the cotyledons and seedling leaves of Cr3529, indicating that the reduced light-harvesting complex 2 accumulation in thylakoid membrane of Cr3529 was due to the decrease of the related gene mRNA level for translation.     

肝细胞癌高表达基因cDNA文库的构建及生物信息学分析

目的:构建肝细胞癌的高表达基因cDNA文库并进行相应的生物信息学分析。方法:应用抑制性消减杂交(SSH)技术,以7对肝癌组织和癌旁组织为实验材料,构建肝癌高表达基因的cDNA文库;使用BioEdit、BLAST和EGAD等软件进行生物信息学分析。结果:获得1450个白色阳性克隆,经PCR扩增后均有100～1000bb的插入片段,经杂交验证选取125个克隆进行测序;经BLAST、EGAD等生物信息学工具分析获得基因83个,其中细胞分裂相关基因5个,参与细胞信号转导或通讯的基因5个,参与细胞结构或运动的基因7个,细胞或器官防御相关基因11个,参与细胞内基因转录或蛋白表达的基因22个,代谢相关基因18个,另有15个未归类基因。结论:利用SSH技术可构建肝细胞癌组织差异表达基因的消减cDNA文库,为进一步深入探讨肝癌的诊断、治疗和预后评估等提供更多的分子指标。     

抑制消减杂交和辐射小鼠血虚模型筛选"四物汤"诱导表达的基因

应用抑制性消减杂交技术构建受辐射小鼠血虚模型在中药四物汤诱导前后消减cDNA库,并从中克隆鉴定出与四物汤药效相关基因。从受辐射小鼠四物汤诱导前后骨髓细胞中提取mRNA并合成cDNA,分别作为tester和driver进行消减杂交及抑制性PCR,将PCR产物与载体连接构建cDNA库,高效电转化大肠杆菌进行库扩增,随机挑取其中的克隆进行酶切、测序分析。成功构建了具有高消减效率的受辐射小鼠四物汤诱导前后的消减cDNA库。库挑选得到512个阳性克隆。随机挑取30个插入片段测序,生物信息学分析结果显示大部分与红细胞分化、骨髓组织修复、结构重建有密切关系,其中8个为新基因片段。应用抑制性消减杂交技术所构建的受辐射小鼠四物汤诱导前后cDNA消减库为大批量筛选、克隆四物汤药效特异性相关基因奠定了基础。并从分子水平上发现四物汤对抑制凋亡、促进骨髓组织修复和结构重建、诱导红细胞分化的基因具有调节作用,可能和四物汤补血作用有关。     

链状亚历山大藻生长衰亡相关基因的筛选

采用抑制性消减杂交(Suppression subtractive hybridization,SSH)技术构建了衰亡期的链状亚历山大藻(Alexandriumcatenella)特异表达的cDNA文库。共获得800个克隆,利用巢式引物进行PCR筛选,最终确定阳性克隆556个。利用斑点杂交技术对这些阳性克隆进行差异筛选,获得差异表达克隆160个。测序后得到片段125个,经过归类,获得21种序列,其中6种在NCBI中经Blast,获得功能基因与其匹配,这些功能基因分别是CHK1类似检测点蛋白(checkpoint-like protein)、核酸外切酶复合体、谷氧还蛋白、Na+/K+ATPase、叶绿体中的一个开放阅读框和pG1蛋白,其他blast无同源序列,可能为新基因。推测链状亚历山大藻的衰亡过程可能涉及到DNA损伤、mRNA降解过程、氧化还原状态的变化、离子动态平衡的变化以及叶绿体一些生理状况的变化等过程。     

Isolation and Identification of Submergence-induced Genes in Maize Seedlings by Suppression Subtractive Hybridization

To investigate the expression profile of maize genes induced by submergence, a subtracted cDNA library of maize seedling roots was constructed using suppression subtractive hybridization (SSH). The cDNA of maize seedling roots treated with submergence (ST) was used as tester and what from untreated roots (UT) as driver. Products of the secondary PCR from the forward subtraction were cloned into T/A vector and transferred into Escherichia coli strain JM10B by electroporation. Four hundred and eight randomly chosen transformants carrying cDNA fragments were screened with PCR-Select Deferential Screening Kit. One hundred and eighty-four cDNA clones were identified as submergence specifically induced or highly expressed. After sequencing and removing redundant cDNAs, we got 95 submergence-induced cDNA clones. Of the 95 cDNA clones, 68 contain the regions with 60%-90% identity to their homolog in GenBank, 21 are expected to be novel genes, only 6 correspond to the published maize sequences. Key words: maize; expression profile; suppression subtractive hybridization (SSH); submergence     

利用SSH方法筛选与鉴定AC3基因缺失小鼠主要嗅觉表皮内的差异表达基因

腺苷酸环化酶3(Adenylate cyclase 3, AC3)基因在小鼠主要嗅觉表皮(Main olfactory epithelium, MOE)内的嗅觉信号传导中起着重要作用, AC3缺失是否会导致MOE内与之相关的基因发生差异表达, 尚待确定。文章利用抑制性消减杂交(Suppression subtractive hybridization, SSH)方法, 以AC3敲除(AC3^-/-)及其同窝出生的野生型(AC3^+/+)小鼠MOE为材料, 构建了正向和反向两个消减文库, 采用斑点杂交对消减文库进行初步筛选, 对筛选出的差异表达基因进行序列测定及生物信息学分析, 并利用荧光定量PCR(qRT-PCR)方法对其进行验证。斑点杂交筛选获得了386个差异表达克隆, 随机选取其中的80个进行DNA序列测定, 经序列比对后发现有62个在GenBank上获得了与之相匹配的基因信息, 其中24个上调差异表达克隆对应于kcnk3、mapk7、megf11等基因, 38个下调差异表达克隆对应于tmem88b、c-mip、skp1a、mlycd等基因。利用Gene Ontology(GO)方法对这些差异表达基因进行蛋白功能注释, 发现它们主要集中在分子结合、细胞周期、生物和细胞过程等功能方面。选取其中上调基因kcnk3和下调基因c-mip、mlycd、tmem88b及trappc5进行qRT-PCR验证。结果表明, 在AC3^-/-小鼠MOE内kcnk3的表达量显著上调, 是对照组小鼠的1.27倍, 而c-mip、mlycd、tmem88b和trappc5的表达量显著下调, 为对照组小鼠的20%、7%、32%和29%。这些基因的功能与K⁺通道、细胞发育与分化、脂肪代谢和膜蛋白转运等密切相关。推测它们可能与AC3基因共同作用, 调节小鼠MOE内的嗅觉信号传导信息。     

Expression patterns in soybean resistant to Phakopsora pachyrhizi reveal the importance of peroxidases and lipoxygenases

Soybean rust caused by Phakopsora pachyrhizi Sydow is a devastating foliar disease that has spread to most soybean growing regions throughout the world, including the USA. Four independent rust resistance genes, Rpp1–Rpp4, have been identified in soybean that recognize specific isolates of P. pachyrhizi. A suppressive subtraction hybridization (SSH) complementary DNA (cDNA) library was constructed from the soybean accession PI200492, which contains Rpp1, after inoculation with two different isolates of P. pachyrhizi that result in susceptible or immune reactions. Both forward and reverse SSH were performed using cDNA from messenger RNA pooled from 1, 6, 12, 24, and 48 h post-inoculation. A total of 1,728 SSH clones were sequenced and compared to sequences in GenBank for similarity. Microarray analyses were conducted on a custom 7883 soybean-cDNA clone array encompassing all of the soybean-rust SSH clones and expressed sequence tags from four other soybean cDNA libraries. Results of the microarray revealed 558 cDNA clones differentially expressed in the immune reaction. The majority of the upregulated cDNA clones fell into the functional category of defense. In particular, cDNA clones with similarity to peroxidases and lipoxygenases were prevalent. Downregulated cDNA clones included those with similarity to cell-wall-associated protein, such as extensins, proline-rich proteins, and xyloglucan endotransglycosylases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genes newly identified as regulated by glucocorticoids in murine thymocytes

Glucocorticoids induce dramatic biochemical and morphological changes in lymphocytes through an unknown process that requires RNA and protein synthesis. In order to identify genes involved in this response, we previously isolated 11 cDNA clones from the murine WEHI-7TG thymoma cell line that correspond to mRNAs induced by glucocorticoids. We now report the isolation of two new cDNA clones whose gene expression is regulated by glucocorticoids in WEHI-7TG cells. We further characterize the two new cDNA clones, as well as those described previously, by examining the response of each of the corresponding mRNAs to glucocorticoids in murine thymocytes. With the exception of two, all cDNAs correspond to genes that are induced by glucocorticoids in murine thymocytes within 4 h of treatment. We previously identified two of the cDNAs as the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein. We have now identified four additional cDNA clones that correspond to the genes for calmodulin, mitochondrial phosphate carrier protein, immunoglobulin (Ig)-related glycoprotein (GP-70), and the 70 kilodalton autoantigen for Lupus and Graves diseases. Two other cDNA clones represent previously undescribed genes: one shares a high similarity to known sequences for the family of G-protein-coupled receptors and the other to a human placental-specific protein, PP11. Another cDNA appears to contain sequences for an unknown gene and the remnants of a mouse transposon. ETn. The remaining clones represent new, unidentified genes induced by glucocorticoids in murine thymocytes and in the WEHI-7TG cell line.