鼻咽癌上皮细胞株HNE1差异表达基因的分离与鉴定

为了分离鼻咽癌差异表达基因 ,应用抑制性扣除杂交技术 ,在正向抑制性扣除杂交中 ,以鼻咽癌上皮细胞株HNE1cDNA作为检测子 ,以人胚鼻咽上皮细胞cDNA作为驱赶子 ;在反向抑制性扣除杂交中 ,以人胚鼻咽上皮细胞cDNA作为检测子 ,以鼻咽癌上皮细胞株HNE1cDNA作为驱赶子 ,分别通过抑制性扣除杂交 ,构建了鼻咽癌上皮细胞株HNE1表达下调和表达上调的两个扣除cDNA文库 .从鼻咽癌相关的扣除cDNA文库中随机挑取 1 2 0 0个克隆 ,采用菌落PCR扩增其插入cDNA片段 ,自动点膜制备成cDNA微阵列膜 ,分别用鼻咽癌上皮细胞株HNE1、人胚鼻咽上皮mRNA经逆转录标记cDNA探针 ,分别与cDNA微阵列膜杂交 ,通过杂交信号的自动扫描分析 ,对杂交信号存在 5倍差异的克隆进行测序 ,获得了 1 0个鼻咽癌差异表达基因的cDNA片段 ,其中 3个为新基因序列 ,其GenBank登录号为 :AF5 1 0 1 88、AF5 1 0 1 89和AF5 1 0 1 90 ,7个代表已知基因序列 .采用RT PCR证实S1 0 0A8,CK1 9和RBP1基因在人胚鼻咽上皮中高表达而在鼻咽癌细胞株HNE1中低表达 .这些结果显示上述基因可能是鼻咽癌发生的重要因素     

Efficacy of SSH PCR in isolating differentially expressed genes

Background  

Suppression Subtractive Hybridization PCR (SSH PCR) is a sophisticated cDNA subtraction method to enrich and isolate differentially expressed genes. Despite its popularity, the method has not been thoroughly studied for its practical efficacy and potential limitations.     

Prokaryotic Suppression Subtractive Hybridization PCR cDNA Subtraction, a Targeted Method To Identify Differentially Expressed Genes

Molecular biology tools can be used to monitor and optimize biological treatment systems, but the application of nucleic acid-based tools has been hindered by the lack of available sequences for environmentally relevant biodegradation genes. The objective of our work was to extend an existing molecular method for eukaryotes to prokaryotes, allowing us to rapidly identify differentially expressed genes for subsequent sequencing. Suppression subtractive hybridization (SSH) PCR cDNA subtraction is a technique that can be used to identify genes that are expressed under specific conditions (e.g., growth on a given pollutant). While excellent methods for eukaryotic SSH PCR cDNA subtraction are available, to our knowledge, no methods previously existed for prokaryotes. This work describes our methodology for prokaryotic SSH PCR cDNA subtraction, which we validated using a model system: Pseudomonas putida mt-2 degrading toluene. cDNA from P. putida mt-2 grown on toluene (model pollutant) or acetate (control substrate) was subjected to our prokaryotic SSH PCR cDNA subtraction protocol to generate subtraction clone libraries. Over 90% of the sequenced clones contained gene fragments encoding toluene-related enzymes, and 20 distinct toluene-related genes from three key operons were sequenced. Based on these results, prokaryotic SSH PCR cDNA subtraction shows promise as a targeted method for gene identification.     

Identification of genes differentially expressed in benign prostatic hyperplasia.

Differences between benign prostatic hyperplasia (BPH) and normal prostate tissue at the level of mRNA expression provide an opportunity to identify candidate genes for this disease. A cDNA subtraction procedure was used to isolate differentially expressed genes in BPH. The subtraction was done by solution hybridization of BPH cDNA against excess normal prostate cDNA. We identified known, EST, and novel genes by sequence and database analysis of the subtracted cDNAs. Several of these cDNAs were used as probes in Northern blotting analysis to confirm over-expression of their corresponding mRNAs in BPH tissues. One highly upregulated sequence of interest shared identity with a known mRNA encoding human NELL2, a protein containing epidermal growth factor-like domains. NELL2 was not previously reported to be expressed in prostate and may code for a novel prostatic growth factor. In situ hybridization analysis of hyperplastic prostate specimens demonstrated that NELL2 mRNA expression is predominantly localized in basal cells of the epithelium. Disease-related changes in the levels of NELL2 may contribute to alterations in epithelial-stromal homeostasis in BPH. (J Histochem Cytochem 49:669-670, 2001)     

A modified cDNA subtraction to identify differentially expressed genes from plants with universal application to other eukaryotes

We have designed a simple and efficient polymerase chain reaction (PCR)-based cDNA subtraction protocol for high-throughput cloning of differentially expressed genes from plants that can be applied to any experimental system and as an alternative to DNA chip technology. Sequence-independent PCR-amplifiable first-strand cDNA population was synthesized by priming oligo-dT primer with a defined 5' heel sequence and ligating another specified single-stranded oligonucleotide primer on the 3' ends of first-strand cDNAs by T4 RNA ligase. A biotin label was introduced into the sense strands of cDNA that must be subtracted by using 5' biotinylated forward primer during PCR amplification to immobilize the sense strand onto the streptavidin-linked paramagnetic beads. The unamplified first strand (antisense) of the interrogating cDNA population was hybridized with a large excess of amplified sense strands of control cDNA. We used magnetic bead technology for the efficient removal of common cDNA population after hybridization to reduce the complexity of the cDNA prior to PCR amplification for the enrichment and sequence abundance normalization of differentially expressed genes. Construction of a subtracted and normalized cDNA library efficiently eliminates common abundant cDNA messages and also increases the probability of identifying clones differentially expressed in low-abundance cDNA messages. We used this method to successfully isolate differentially expressed genes from Pennisetum seedlings in response to salinity stress. Sequence analysis of the selected clones showed homologies to genes that were reported previously and shown to be involved in plant stress adaptation.     

Generation of full-length cDNA libraries enriched for differentially expressed genes for functional genomics

Here, we describe the application of a RecA-based cloning technology to generate full-length cDNA libraries enriched for genes that are differentially expressed between tumor and normal tissue samples. First, we show that the RecA-based method can be used to enrich cDNA libraries for several target genes in a single reaction. Then, we demonstrate that this method can be extended to enrich a cDNA library for many full-length cDNA clones using fragments derived from a subtracted cDNA population. The results of these studies show that this RecA-mediated cloning technology can be used to convert subtracted cDNAs or a mixture of several cDNA fragments corresponding to differentially expressed genes into a full-length library in a single reaction. This procedure yields a population of expression-ready clones that can be used for further high-throughput functional screening.     

Isolation of diallyl trisulfide inducible differentially expressed genes in human gastric cancer cells by modified cDNA representational difference analysis

Extensive epidemiologic studies indicated protective effects of consumption of garlic on reducing human gastric cancer (HGC) incidence. Diallyl trisulfide (DATS), a critical organic allyl sulfur component of garlic, was reported to have chemopreventive effects in inhibiting tumor process. We used DATS to treat HGC cell line BGC823 cells, and showed that DATS induces G1/S arrest and apoptosis in BGC823 cells demonstrated by a flow cytometric analysis. To further isolate DATS inducible differentially expressed genes in BGC823 cells, we combined a highly specific subtractive hybridization of cDNA representational difference analysis (cDNA RDA) with a sensitive bidirectional radioactive detection of mRNA differential display (mRNA DD) to develop a subtractive hybridization differential display (SHDD) method. This modified method adopted a first round of bidirectional subtractive hybridization between two sample cDNAs and a second round of bidirectional subtractive hybridization between the two resultant first-round difference products. Bidirectional subtractive hybridizations magnified the differences between the two sample cDNAs and favored isolating mRNA species with very small expression differences. We employed the SHDD method to detect DATS inducible differentially expressed genes in BGC823 cells. A total of 14 cDNA fragments (11 upregulated and 3 downregulated by DATS treatment) were isolated and confirmed by reverse Northern blot analysis. Our data show that SHDD is a powerful technique for identifying differentially expressed mRNA species between two sample cDNAs and provide useful cellular and molecular information for understanding the effects of garlic against human gastric cancer.     

一种简单快速克隆IL-6信号转导相关基因的方法

细胞因子作用于受体时的一个重要结果是诱导基因表达。为了克隆与IL-6诱导相关的基因,我们利用一个快速的改良DD-PCR方法,分离并检测了IL-6诱导和未诱导的U937细胞的差异表达基因。用三个完全变性的6—mer引物进行反转录,用2或3个较长的随机引物进行PCR扩增,扩增产物很在2%琼脂糖凝胶电泳上分离,之后回收差异片段并直接用于克隆和测序。在研究中,获得了7个不同的EST,序列分析表明其中2个EST可能是与细胞信号转导相关的新基因片段;反向Northern杂交证实它们是与IL-6作用相关的差异表达基因。     

Full-Length Normalization Subtractive Hybridization: A Novel Method for Generating Differentially Expressed cDNAs

Here we present a novel method termed full-length normalization subtractive hybridization (FNSH) for efficiently generating subtracted cDNA libraries with a high degree of productivity. This method has the ability to isolate full-length differentially expressed genes from target samples. Normalization and subtraction of FNSH are performed simultaneously with efficiency equal to or even higher than that of suppression subtractive hybridization. Using FNSH, we have isolated at least 40 unique cDNAs that are expressed in terminal ampullae but not in the ovaries of the prawn Macrobrachium rosenbergii from 120 randomly picked subtracted clones. Sequence analysis shows that 37 of the 40 cDNAs are full length.     

A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester–driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research.     

Use of SMART-generated cDNA for gene expression studies in multiple human tumors

We demonstrate here that SMART PCR-amplified cDNAs arrayed on a nylon membrane are suitable for high-throughput tissue expression profiling when starting biological materials are limited. We show that SMART cDNA accurately reflects gene expression patterns found in total RNA by comparing the expression level of several target genes in SMART PCR-amplified cDNAs and their corresponding total RNAs. We also arrayed cDNAs from 68 matched tumor and normal samples on a nylon membrane to determine whether SMART PCR-amplified cDNA could be used for detecting differentially expressed genes in these tissues. These arrays containing normalized tumor and normal cDNAs were hybridized with probes for glutathione peroxidase and gelsolin. The hybridization results revealed cancer-related and patient-specific gene expression differences between tumor and normal tissues for these genes. These studies show that SMART PCR-amplified cDNAs maintain the complexity of the original mRNA population and are thus suitable for high-throughput studies to compare the relative abundance of target genes and to detect differentially expressed genes in a wide variety of tissues simultaneously.     

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     

抑制差减杂交法分离玉米幼苗淹水诱导表达基因

以淹水处理（submergence-treated,ST)的玉米（Zea maysL.)幼苗根部cDNA为目标群体,未处理（untreated,UT)的玉米幼苗cDNA为对照群体,进行抑制差减杂交。用经过UT差减的STcDNA构建了一个含有大约2000个独立克隆的差减文库。对随机挑取的408个克隆进行差异筛选。获得了184个在ST中特异表达或表达增强的候选克隆。对其中155个cDNA克隆测序并去除重复克隆后,共得到95个差异表达的cDNA片段。GenBank中BLAST查询结果表明;6个克隆为已知的玉米核苷酸序列;68个克隆与已知基因或EST序列部分区域的同源性为60%-90%;21个克隆在GenBank中无法查到对应的同源序列。可能代表了新基因。或者由于序列位于变异丰富的3′端而无法查到与其他物种基因的同源性。     

Isolation of novel human fetal brain cDNAs mapped to human chromosome bands, 1q25 and 8q24.1

We isolated chromosome band-specific human fetal brain cDNAs by the microdissection mediated cDNA capture method, and localized these cDNA using in situ hybridization histochemistry with developing rat brain sections. Uni-Amp cDNAs were prepared from an 18-week old human fetal brain, and hybridized to human metaphase chromosomes. Eight Uni-Amp cDNAs, hybridized to chromosome band 1q25 or 8q24.1, were recovered by microdissection and PCR amplification with Uni-Amp primers. Among these cDNAs, two novel genes (FB113 of 8q24.1 and FB134 of 1q25) showed a temporospatially interesting expression pattern in the developing rat brains. The expression of FB113 was under dynamic regulation in the developing granule cells of cerebellum and dentate gyrus. FB134 showed a nervous tissue specific expression pattern and an exclusively prominent expression in the developing presubiculum and parasubiculum. By the fluorescence in situ hybridization using human genomic DNAs, FB113 and FB134 were mapped back to the human chromosome bands 8q24.1 and 1q25, respectively. These results indicate that combined application of the microdissection mediated cDNA capture method and in situ hybridization histochemistry can be used for the isolation of chromosomal band-specific genes related to brain development or human genetic diseases.     

Applying genomics to the avian inner ear: development of subtractive cDNA resources for exploring sensory function and hair cell regeneration

We applied a micro-cDNA-based subtraction method to identify genes expressed in the regenerating sensory epithelia (SE) of the chicken inner ear. Sensory hair cells in the avian utricle SE are in a constant state of turnover, where dying hair cells are replaced by new ones derived from supporting cells. In contrast, hair cells in the cochlea remain quiescent unless damaged. We used this difference to enrich for utricle-specific genes, using reiterative cDNA subtraction and demonstrate enrichment for utricle-specific sequences. A total of 1710 cDNA sequence reads revealed the presence of many cDNAs encoding known structural components of the SE (for example, Harmonin and beta-tectorin), proteins involved in cellular proliferation, such as P311, HIPK2, and SPALT1, among many others of unknown function. These libraries are the first of their kind and should prove useful for the discovery of candidate genes for hearing disorders, regenerative and apoptotic pathways, and novel chicken ESTs.