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.     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

Gene expression in mouse oocytes and preimplantation embryos: use of suppression subtractive hybridization to identify oocyte- and embryo-specific genes

The paucity of biological material has inhibited identifying genes that are differentially expressed during mammalian oogenesis and preimplantation development. We report here the linear amplification of mRNA from small numbers of mouse oocytes and preimplantation embryos to generate amounts of sense RNA that are sufficient for suppression subtractive hybridization. The resulting oocyte-specific and 8-cell-specific cDNA libraries were partially characterized, and the known oocyte-specific ZP1, ZP2, GDF-9, BMP15, and H1(oo) genes were found in the oocyte-specific cDNA library but not in the 8-cell-specific library. Further characterization of the subtracted oocyte and 8-cell embryo cDNA libraries should furnish a trove of information regarding temporal changes in gene expression during oogenesis and preimplantation development in the mouse.     

Identification of fungal (Magnaporthe grisea) stress-induced genes in wild rice (Oryza minuta)

To identify fungal stress-related genes in wild rice, Oryza minuta, we constructed a subtracted library using suppression subtractive hybridization in combination with mirror orientation selection. DNA chips containing 960 randomly selected cDNA clones were applied by reverse Northern analysis to eliminate false positive clones from the library and to prescreen differentially expressed genes. In total, 377 cDNA clones were selected on the basis of their signal intensities and expression ratios. Sequence analyses of these 377 cDNA fragments revealed that 180 of them (47.7%) represented unique genes. Of these180 cDNAs, 89 clones (49.6%) showed significant homologies to previously known genes, while the remaining 91 did not match any known sequences. The putative functions of the 180 unique ESTs were categorized by aligning them with MIPS data. They were classified into seven different groups using microarray data-derived expression patterns and verified by Northern blotting.Abbreviations ER: Endoplasmic reticulum - EST: Expressed sequence tag - MIPS: Munich Information Center for Protein Sequences - MOS: Mirror orientation selection - NCBI: National Center for Biotechnology Information - omfi: Oryza minuta fungal-stress induced - PCD: Programmed cell death - PDI: Proteins disulfide isomerase - SSH: Suppression subtractive hybridization Communicated by I.S. ChungK.S. Shim and S.K. Cho contributed equally to this work.     

Subtractive screening of genes involved in cellular senescence

We attempted to identify the genes involved in cellularsenescence, telomere maintenance and telomerase regulationthrough subtractive screening of cDNA libraries prepared froma human lung adenocarcinoma cell line A549 and its sublinesnamed A5DC7, CK and AST-9. Cell phenotypes of A5DC7, CK andAST-9 are normal cell-like, cancer cell-like and intermediate,respectively. These cell lines have different phenotypes interms of telomerase activity and telomere maintenance, andthus are thought to be useful for identifying the genesinvolved in cellular senescence and telomerase regulation. In this study, we identified 86 independent cDNA clones bysubtractive screening. Among these cDNA clones, subtractingA5DC7 cDNAs from A549 cDNAs and CK cDNAs gave 7 and 3 cDNAclones which highly and specifically expressed in tester celllines. Genes corresponding to these 10 cDNA clones mightparticipate in maintaining cancer-cell phenotypes. As aresult of database searching, each four of A549 specific cDNAclones are found to correspond to known cDNAs. Each two ofA549 specific and two of CK specific cDNA clones have highhomology to independent ESTs. Sequences having homology toeach one of A549 specific and one of CK specific cDNA cloneshave not been deposited in the Genbank database, indicatingthat these two cDNA clones are part of novel genes. Weanticipate that their involvement in telomerase regulationand/or senescence program can be clarified by functionalanalysis using each full-length cDNA.     

Identification of cAMP analogue inducible genes in RAW264 macrophages

RNA was isolated from RAW264 cells treated with or without 8-Br-cAMP and the differential display and subtractive hybridization methods were performed. One hundred and twenty-five differentially displayed bands were identified. Upon Northern blot analysis, only three of these bands were confirmed as cAMP inducible mRNAs, named cI-1, cI-2, and cI-3 (for cAMP inducible genes 1-3). The cI-3 probe was identical to a previously known gene, gly96. Using the novel cI-1 and cI-2 partial cDNAs as probes, a mouse macrophage cDNA library was screened and the two full length genes were cloned, sequenced, and characterized as encoding large hydrophobic proteins. One hundred and fifteen partial cDNA clones from a subtractive hybridization library were also screened by Northern blot and 64 were found to be cAMP inducible. Of these, 45 represented 31 known unique genes in the GenBank nr database (cI-4-34), and 19 clones representing 15 unique sequences were not in the nr database (cI-35-49). One of the previously known genes was ABC1, the Tangier disease gene, which was identified from four independent partial cDNAs. ABC1 was upregulated in RAW cells by cAMP, concurrent with the cAMP induction of lipid efflux to apolipoprotein A1.     

Identification of novel and known ovary-specific genes including ZP2, in a marsupial, the stripe-faced dunnart

During the early stages of oogenesis, oocyte-specific factors, synthesized by and stored within the oocyte, play critical roles during oogenesis, folliculogenesis, fertilization and early embryonic development in the mouse. The identification of marsupial maternal factors, expressed specifically in the ovary or oocyte, may provide an insight into the conserved evolutionary mechanisms that drive mammalian oocyte development to cleavage stages. In this study, 10 clones including dunnart ZP2 and c-mos, isolated by cDNA representational difference analysis, were validated by RT-PCR for ovary-specific expression. This novel combination of techniques to isolate ovary-specific genes has identified three novel genes with ovary-specific expression. Both dunnart ZP2 and c-mos exhibited ovary-specific expression, making this study the first isolation of c-mos in a marsupial species. Dunnart ZP2 expression was examined in detail by in situ hybridization and results indicate oocyte-specific expression of dunnart ZP2 in the cytoplasm of oocytes of primordial, primary and secondary follicles with expression being highest in oocytes of primary follicles. ZP2 was not expressed in granulosa cells of any follicles.     

Large-scale analysis of differential gene expression in the hindlimb muscles and diaphragm of mdx mouse

The mdx mouse is an animal model for Duchenne muscular dystrophy (DMD), which is caused by the absence of dystrophin. Mdx limb muscles substantially compensate for the lack of dystrophin while the diaphragm is affected like DMD skeletal muscles. To understand better the complex cascade of molecular events leading to muscle degeneration and compensatory processes in mdx muscles, we analyzed alterations of gene expression in mdx hindlimb and diaphragm muscles as compared to their normal counterparts. The strategy was based on suppression subtractive hybridization followed by reverse Northern quantitative hybridization. Four subtracted/normalized libraries, containing cDNA clones up- or downregulated in mdx hindlimb muscles or diaphragm, were constructed and a total of 1536 cDNA clones were analyzed. Ninety-three cDNAs were found to be differentially expressed in mdx hindlimb muscles and/or diaphragm. They corresponded to 54 known genes and 39 novel cDNAs. The potential role of the known genes is discussed in the context of the mdx phenotype.     

Differential gene expression in fully-grown oocytes between gynogenetic and gonochoristic crucian carps.

Silver crucian carp (Carassius auratus gibelio) is a unique triploid bisexual species that can reproduce by gynogenesis. As all other gynogenetic animals, it keeps its chromosome integrity by inhibiting the first meiosis division (no extrusion of the first pole body). To understand the molecular events governing this reproduction mode, suppression subtractive hybridization was used to identify the genes differentially expressed in fully-grown oocytes of the gynogenetic and gonochoristic crucian carp (gyno-carp and gono-carp). From two specific subtractive cDNA libraries, the clones screened out by dot blots and virtual Northern blots were chosen to clone full-length cDNA by RACE. Four differentially expressed genes were obtained. Two are novel genes and are expressed specifically in the oocytes. The gyno-carp stores much more mRNA of cyclin A2, a new member of the fish A-type cyclin gene, in its fully-grown oocyte than in the gono-carp. The last gene is histone H2A. The histone H2As of these two closely related crucian carps are quite different in the C-terminus. Preliminary characterization of the four genes has been analyzed by nucleotide and deduced amino acid sequence and Northern analysis.     

红曲菌cDNA消减文库的构建

应用抑制性消减杂交技术,构建红曲菌产桔霉素和不产桔霉素差异表达的cDNA消减文库.分别从产桔霉素和不产桔霉素的红曲菌丝体中提取mRNA,依次合成单链和双链cDNA,经酶切成大小为250～750bp的片断,将产桔霉素的cDNA分为两组,分别与两种不同的接头连接,再与不产桔霉素的红曲菌的cDNA进行两次消减杂交及两次抑制性PCR后,将产物与T/A载体连接构建成功cDNA消减文库,并转染大肠杆菌进行文库扩增,文库扩增后得到283个克隆,经PCR法快速测定,均得到250～750bp的插入片断.所构建的红曲菌cDNA消减文库为进一步筛选红曲菌中与产桔霉素性状相关的基因奠定了基础.     

A mammalian oocyte-specific linker histone gene H1oo: homology with the genes for the oocyte-specific cleavage stage histone (cs-H1) of sea urchin and the B4/H1M histone of the frog

Oocytes and early embryos of multiple (non-mammalian) species lack the somatic form of the linker histone H1. To the best of our knowledge, a mammalian oocyte-specific linker (H1) histone(s) has not, as yet, been reported. We have uncovered the cDNA in question in the course of a differential screening (suppression subtractive hybridization (SSH)) project. Elucidation of the full-length sequence of this novel 1.2 kb cDNA led to the identification of a 912 bp open reading frame. The latter encoded a novel 34 kDa linker histone protein comprised of 304 amino acids, tentatively named H1oo. Amino acid BLAST analysis revealed that H1oo displayed the highest sequence homology to the oocyte-specific B4 histone of the frog, the respective central globular (putative DNA binding) domains displaying 54% identity. Substantial homology to the cs-H1 protein of the sea urchin oocyte was also apparent. While most oocytic mRNAs corresponding to somatic linker histones are not polyadenylated (and remain untranslated), the mRNAs of (non-mammalian) oocyte-specific linker histones and of mammalian H1oo, are polyadenylated, a process driven by the consensus signal sequence, AAUAAA, detected in the 3'-untranslated region of the H1oo cDNA. Our data suggest that the mouse oocyte-specific linker histone H1oo (1) constitutes a novel mammalian homolog of the oocyte-specific linker histone B4 of the frog and of the cs-H1 linker histone of the sea urchin; (2) is expressed as early as the GV (PI) stage oocyte, persisting into the MII stage oocyte, the oocytic polar bodies, and the two-cell embryo, extinction becoming apparent at the four- to eight-cell embryonic stage; and (3) may play a key role in the control of gene expression during oogenesis and early embryogenesis, presumably through the perturbation of chromatin structure.     

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     

Prediction of the coding sequences of mouse homologues of KIAA gene: II. The complete nucleotide sequences of 400 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have accumulated information of the coding sequences of uncharacterized human genes, which are known as KIAA genes, and the number of these genes exceeds 2000 at present. As an extension of this sequencing project, we recently have begun to accumulate mouse KIAA-homologous cDNAs, because it would be useful to prepare a set of human and mouse homologous cDNA pairs for further functional analysis of the KIAA genes. We herein present the entire sequences of 400 mouse KIAA cDNA clones and 4 novel cDNA clones which were incidentally identified during this project. Most of clones entirely sequenced in this study were selected by computer-assisted analysis of terminal sequences of the cDNAs. The average size of the 404 cDNA sequences reached 5.3 kb and that of the deduced amino acid sequences from these cDNAs was 868 amino acid residues. The results of sequence analyses of these clones showed that single mouse KIAA cDNAs bridged two different human KIAA cDNAs in some cases, which indicated that these two human KIAA cDNAs were derived from single genes although they had been supposed to originate from different genes. Furthermore, we successfully mapped all the mouse KIAA cDNAs along the genome using a recently published mouse genome draft sequence.     

Prediction of the coding sequences of mouse homologues of KIAA gene: III. the complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have conducted a human cDNA project to predict protein-coding sequences (CDSs) in large cDNAs (> 4 kb) since 1994, and the number of newly identified genes, known as KIAA genes, already exceeds 2000. The ultimate goal of this project is to clarify the physiological functions of the proteins encoded by KIAA genes. To this end, the project has recently been expanded to include isolation and characterization of mouse KIAA-counterpart genes. We herein present the entire sequences and the chromosome loci of 500 mKIAA cDNA clones and 13 novel cDNA clones that were incidentally identified during this project. The average size of the 513 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 816 amino acid residues. By comparison of the predicted CDSs between mouse and human KIAAs, 12 mKIAA cDNA clones were assumed to be differently spliced isoforms of the human cDNA clones. The comparison of mouse and human sequences also revealed that four pairs of human KIAA cDNAs are derived from single genes. Notably, a homology search against the public database indicated that 4 out of 13 novel cDNA clones were homologous to the disease-related genes.     

Screening and comparison of differentially expressed genes between one MDR-TB strain and the virulent M. tuberculosis H37Rv

To screen and compare the differentially expressed genes between one MDR-TB strain separated from one child patient and the virulent Mycobacterium tuberculosis H37Rv, suppression subtractive hybridization (SSH) technology was used to build a library of cDNAs that were differentially expressed in the MDR and H37Rv. From this cDNA library, genes that were expressed in the MDR-TB but not in the H37Rv were selected for gene sequencing and homology analysis; 113 positive clones were obtained, their cDNA fragments were sequenced, and homology analysis was performed. Four novel sequences were identified. The results provide a partial list of genes differentially expressed in MDR-TB and four novel genes were found. Identification of these genes may contribute to our understanding of MDR-TB development.