Reverse Subtracted cDNA Libraries Research Articles

Differentially-expressed genes identified by suppression subtractive hybridization in the bone marrow hematopoietic stem cells of patients with psoriasis.

Psoriasis is a T cell-mediated, chronic, relapsing and inflammatory cutaneous disorder. The dysfunctional activity of T cells in patients with psoriasis is attributed to bone marrow hematopoietic stem cells (BMHSCs). To understand the pathogenic roles of BMHSCs in psoriasis, a differential gene expression analysis was performed using suppression subtractive hybridization of the BMHSCs from a patient with psoriasis and a healthy control. Using a cDNA array dot blot screening to screen 600 genes from forward- and reverse-subtracted cDNA libraries, 17 differentially-expressed sequence tags (ESTs) were identified. The genes within the ESTs were observed to be the homologs of genes that are involved in various cellular processes, including hormone signaling, RNA catabolism, protein ADP DNA base melting, transcriptional regulation, cell cycle regulation and metabolism. CD45, which was overexpressed in the psoriatic BMHSCs, was further analyzed using relative quantitative polymerase chain reaction. In addition, the levels of CD45 in the peripheral blood cells (PBCs) of the patients with psoriasis were markedly increased and closely associated with disease severity. An abnormality of hematopoietic progenitor cells, e.g., CD45 overexpression, may be transferred to PBCs via hematopoiesis, and may account for the psoriasis-inducing properties of activated T cells.

Screening and identification of lung cancer metastasis-related genes by suppression subtractive hybridization.

Lung cancer metastasis is a complicated process in which multiple stages and multiple genes are involved. There is an urgent need to use new molecular biology techniques to get more systematic information and have a general idea of the molecular events that take place in lung cancer metastasis. The object of this study was to construct the subtracted cDNA libraries of different metastatic potential lung cancer cell lines, NL9980 and L9981, which were established and screened from human lung large cell carcinoma cell line, WCQH-9801. The forward and reverse subtracted cDNA libraries were constructed in the large cell lung cancer cell lines NL9980 and L9981 with the same heredity background but different metastatic potential, by suppression subtractive hybridization (SSH). The positive clones were preliminarily screened by blue-white colony and precisely identified by PCR. The forward and reverse subtracted libraries were screened and identified by dot blot so as to obtain the clones corresponding to gene segments with differential expression. DNA sequencing was performed to analyze the sequences of differential expression segments, which were then searched and compared using the Basic Local Alignment Search Tool from The National Center for Biotechnology Information NCBI BLAST tools. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) and western blotting were performed to confirm the differential expressed genes both on RNA and protein levels. The forward and reverse subtracted cDNA libraries of the different large cell lung cancer cell lines with metastatic potential were successfully constructed. With blue-white colony and dot blot, 307 positive clones in the forward subtracted library and 78 positive clones in the reverse subtracted library were obtained. Fifty-five clones were successfully sequenced in the forward subtracted library while 31 clones were successfully sequenced in the reverse subtracted library. One new expressed sequence tag (EST) segment was identified from the reverse subtracted cDNA library and was successfully submitted to GenBank and embodied by GenBank. For the differentially expressed genes between L9981 and NL9980 screened by SSH, four genes, ANXA2, KRT18, ACTG1 was upregulated in L9981 cells compared to NL9980 cells. Annexin A2 (which was encoded by ANXA2), γ-actin (which was encoded by ACTG1), and aldose reductase (which was encoded by AKR1B1) proteins were upregulated in L9981 cells compared to NL9980 cells by western blotting. The forward and reverse subtracted cDNA libraries of different metastatic potential large cell lung cancer cell lines were successfully constructed by SSH. A series of genes have been screened out to have significantly different expression levels between lung cancer cell lines NL9980 and L9981. A new EST segment that may represent a new metastasis-related gene has been identified. Consistent with the result of SSH, both quantitative real-time RT-PCR and western Blotting confirmed the upregulation of ANXA2, ACTG1 and AKR1B1 in lung cancer cell line L9981 compared with NL9980. These three genes may play important roles in lung cancer metastasis.

Construction of differentially expressed genes library of bighead carp ( Aristichthys nobilis) exposed to microcystin-lr using ssh and expression profile of related genes

Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria (blue-green algae). There are more than 70 MCs variants of which the most common and widely studied is MC-LR. We screened the hepatocellular differentially expressed genes against MC-LR in the bighead carp ( Aristichthys nobilis). Suppression subtractive hybridization was used to construct the forward subtracted and reverse subtracted cDNA libraries, and one hundred and thirty two positive clones (seventy one in forward library and sixty one in reverse library) were randomly selected and sequenced. Finally, fifty five reliable sequences from the forward subtracted library were used in a homology search by BLASTn and BLASTx, as were 57 reliable sequences from the reverse subtracted library. Furthermore, eight analyzed sequences from the forward subtracted cDNA library and seven from the reverse subtracted library were found to be non-homologous sequences. The screening identified genes induced by MC-LR in both libraries that are involved in various processes, such as energy metabolism, immunity, and apoptosis. Some are cytoskeleton- and transportation-related genes, while signal transduction-related genes were also found. Significant genes, such as the apoptosis-related gene p53 and the proto-oncogene c-myc, are involved in inhibition of the MC-LR response in the reverse subtracted library. In addition, several immune-related genes, which play an important role in antioxidation and detoxification of MC-LR, were characterized and identified in both of the subtracted libraries. The study provides the basic data to further identify the genes and molecular mechanism of detoxification of microcystins.

Screening for and identification of differentially expressed genes in hemopoietic stem cells from patients with psoriasis

Objective To investigate the roles of hemopoietic stem cells (HSCs) in the pathogenesis of psoriasis. Methods HSCs were isolated from the bone marrow of a patient with psoriasis vulgaris and a normal human control. Forward- and reverse-subtracted cDNA libraries were constructed by using suppression subtractive hybridization (SSH) technique between HSCs from the patient and control. Bacterial PCR and dot hybridization were performed to screen for positive clones followed by gene sequencing, identification and functional analysis. Real time quantitative PCR was carried out to measure the mRNA expression of interferon-γ and thymosin 10 (TMSB10). Results Nine genes were screened from the forward-subtracted cDNA library which encoded interferon-γ, tyrosine phosphatase, SUMO1 activase, etc, and 8 genes including the TMSB10-encoding gene were screened from the reverse-subtracted cDNA library. The relative expression level of interferon-γ in HSCs from the patient was 47.5 times that in HSCs from the control, while the level of TMSB10 from the control was 22.6 times that from the patient. Conclusion The abnormal expression of 17 genes which encode interferon-γ, thyrosin, and so on, may be involved in the development of psoriasis. Key words: Psoriasis; Bone marrow; Hematopoietic stem cells; Gene expression

Analysis of the Petunia hybrida double flower transcriptome using suppression subtractive hybridization

The aim of this study was to characterize the transcriptional response that is associated with the Petunia double flower phenotype with a view to understanding the underlying molecular mechanisms. We created forward and reverse subtracted cDNA libraries to represent the up-regulated genes found in the double and single flowers obtained from segregated progenies through backcross, respectively. This was achieved using suppression subtractive hybridization (SSH) followed by differential screening through dot blot hybridization. In total, 285 expressed sequence tags (ESTs) were identified. Analysis using Blastx searches showed that the libraries contained several important developmental genes. The expression patterns of selected genes, believed to be involved in the development of Petunia double flowers, were further analyzed by semi-quantitative or real-time reverse transcription-polymerase chain reaction (RT-PCR). These analyses were conducted using RNA extracts taken from specific floral organs and from flowers at various stages of development. Each of the selected genes showed differential expression levels between single and double flowers, and this was particularly clear in young flower buds, thereby suggesting that the genes are important for the early development of the double flower form. In particular, we discuss the expression patterns of genes with putative roles relating to cell expansion, organ fusion and organ number. Other genes, with less well-documented roles, may be involved in modulating the development of the Petunia double flower. Thus, such genes appear to function along with the major regulatory MADS-box genes as part of a complicated network of genetic factors that direct the formation of the Petunia double flower.

Identification of differentially expressed genes in the growth plate of broiler chickens with thiram-induced tibial dyschondroplasia

Tibial dyschondroplasia (TD) is characterized by expansion of the proximal growth plates of the tibiotarsus that fail to form bone, lack blood vessels, and contain non-viable cells. Thiram (a carbamate pesticide), when fed to young broiler chicks, induces TD with high regularity and precision. We used this experimental model to understand the cause of the defects associated with TD by selecting and identifying the genes differentially expressed in the TD growth plate of broiler chickens. Broiler chicks at 7 days of age were randomly divided into two groups. After fasting overnight, they were fed with regular diet (control) or the same diet containing 100 mg/kg thiram for 96 h to induce TD (thiram-fed). mRNA was purified from the growth plates of control and thiram-fed broilers. Forward and reverse-subtracted cDNA libraries were generated by suppression subtractive hybridization technology. Ten selected genes from cDNA libraries were identified by real-time quantitative polymerase chain reaction. All were differentially expressed in TD growth plates (P<0.05 or P<0.01). The levels of collagen type X (Col X), pro-alpha-1 collagen type I (Col I α1), collagen type IX (Col IX), NADH dehydrogenase (NADH DH), cytochrome C oxidase subunit III (COX III), enolase 1, alpha (ENO1), carbonic anhydrase II (CA2) and heat shock protein 90 (Hsp90) mRNA transcripts were up-regulated, while the expression levels of Matrilin 3 (MATN3) and chondromodulin-I (ChM-I) were down-regulated. Col I and Hsp90 were detected by immunohistochemistry at different stages. Given that these genes are involved in matrix formation, endochondral ossification, developmental regulation, electron transport in the mitochondrial respiratory chain and vascularization, our findings may provide new insights into understanding the pathogenesis of TD.

Analysis of gene expression in rice root tips treated with R-1-α-methylbenzyl-3- p-tolylurea using PCR-based suppression subtractive hybridization

The optically active chemical 1-α-methylbenzyl-3- p-tolylurea (MBTU) shows diverse properties in plant physiology. The root growth of Cyperus paddy weeds and plants belonging to the tribe Oryzeae are inhibited enantioselectively by R-MBTU. R-MBTU’s mode of action is not yet known. Using the PCR-based suppression subtractive hybridization (SSH) technique, we aimed to elucidate the mechanisms by which R-MBTU inhibits root growth of rice ( Oryza sativa L .). Forward and reverse subtracted cDNA libraries were obtained after cloning, and these clones were analyzed by quantitative real-time RT-PCR. R-MBTU treatment resulted in down-regulated mRNA levels of several clones; putatively a NADH-dependent glutamate synthase, plastid aspartate aminotransferase, endo-1,4-β-glucanase Cel1, 26S proteasome regulatory subunit S2, and serine/threonine-protein kinase. The mRNA level of a putative histone deacetylase was up-regulated. The putative functions of these clones suggest that root growth inhibition by R-MBTU may be associated with disruption of nitrogen metabolism, amino acid biosynthesis, cellulose synthesis, and cell cycle, and repression of transcription of various genes. We discuss how and which metabolic pathways are affected in the inhibition of root growth with R-MBTU treatment.

Study of Cold Tolerance in Blueberry Using EST Libraries, cDNA Microarrays, and Subtractive Hybridization

To gain a better understanding of changes in gene expression associated with cold acclimation in the woody perennial blueberry (Vaccinium corymbosum L.) and ultimately use this information to develop more freeze-tolerant cultivars, a genomics approach based on the analysis of expressed sequence tags (ESTs) and microarrays was undertaken. Initially, two standard cDNA libraries, constructed using RNA from cold-acclimated (CA) and nonacclimated (NA) floral buds of the blueberry cultivar Bluecrop, were used for the generation of ≈2400 ESTs, half from each library. Putative functions were assigned to cDNAs based on homology to other genes/ESTs from GenBank. From contig analyses, 796 and 865 unique transcripts were identified from the CA and NA libraries, respectively. The most highly abundant cDNAs, that were picked many more times from one library than from the other, were identified as representing potentially differentially expressed transcripts. A cDNA microarray was constructed and used to study gene expression under cold-acclimating conditions in the field and cold room. Results indicated that the abundance of transcripts of numerous blueberry genes change during cold acclimation, including genes not found previously to be cold-responsive in Arabidopsis, and, interestingly, more transcripts were found to be upregulated under cold room conditions than under field conditions. Finally, forward and reverse subtracted cDNA libraries were prepared from ‘Bluecrop’ RNA to enrich for transcripts that are expressed at higher levels in floral buds at 400 h and at 0 h of low-temperature exposure, respectively. Many genes encoding putative transcription factors and other proteins related to signal transduction were identified from both libraries.

Gene expression analysis of germinating rice seeds responding to high hydrostatic pressure

High hydrostatic pressure (HHP) is an extreme thermal-physical stress affecting multiple cellular activities. Recently, we found that HHP treatment caused various physiological changes in rice. To investigate the molecular mechanisms of plant response to HHP, we constructed forward and reverse subtracted cDNA libraries of rice seeds treated with 75MPa hydrostatic pressure for 12h by suppression subtractive hybridization in combination with mirror orientation selection. Of 97 clones isolated through microarray dot-blot and sequenced, 45 were unique genes. Among these 45 unique cDNAs, 29 clones showed significant sequence similarity to known genes, 12 were homologous to genes with unknown function, and the remaining 4 clones did not match any known sequences. Most of the genes with known function were involved in metabolism, defense response, transcriptional regulation, transportation regulation, and signal transduction. To our knowledge, this is the first gene expression analysis of rice in response to HHP. The expression profiles of the genes identified in this study provide useful information regarding molecular processes, including alteration of metabolism and adaptation response caused by HHP.

Identification and analysis of differentially expressed genes in differentiating xylem of Chinese fir (Cunninghamia lanceolata) by suppression subtractive hybridization

Wood is an important raw material for global industries with rapidly increasing demand. To isolate the genes differentially expressed during xylogenesis of Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.), we used a novel system. Forward and reverse subtracted cDNA libraries were constructed using the suppression subtractive hybridization method; for the forward library we used cDNA from the mutant Dugansha as the tester and cDNA from the wild-type clone Jurong 0 as the driver, and for the reverse library we used Jurong 0 cDNA as the tester and Dugansha cDNA as the driver. Transcriptional profiling was performed using a macroarray with 4 digoxigenin-labeled probes. We obtained 618 and 409 clones from the forward and the reverse subtracted library, respectively. A total of 405 unique expressed sequence tags (ESTs) were obtained. Forty percent of the ESTs exhibited homologies with proteins of known function and fell into 4 major classes: metabolism, cell wall biogenesis and remodeling, signal transduction, and stress. Real-time PCR was performed to confirm the results. The expression levels of 11 selected ESTs were consistent with both macroarray and real-time PCR results. The systematic analysis of genes involved in wood formation in Chinese fir provides valuable insights into the molecular mechanisms involved in xylem differentiation and is an important resource for forest research that can be directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

Construction and screening of the subtracted cDNA library of human large cell lung cancer lines with different metastatic potentials

Screening metastatic-related genes of lung cancer is helpful to understand the molecular mechanisms of lung cancer invasion and metastasis. In order to screen the differential expression genes related to metastasis of lung cancer, we constructed and preliminarily screened the subtracted cDNA libraries of human large cell lung cancer cell lines with different metastatic potentials in this study. Subtracted cDNA library was constructed in the different metastastic potential cell lines NL9980 and L9981 by suppression subtractive hybridization (SSH) method. The positive clones were preliminarily screened by blue-white colony based on the α-complementary principal, and precisely identified by PCR. The forward and reverse subtracted libraries were screened and identified by dot blot to obtain the clones corresponding to differential expression segments. The subtracted cDNA libraries were successfully constructed in the different metastastic potential cell lines NL9980 and L9981. Three hundred and seven positive clones in the forward subtracted library and 78 positive clones in the reverse subtracted library were obtained by the dot blot method. SSH is proved to be an efficient tool for differential expression gene cloning. The forward and reverse subtracted cDNA libraries of different metastastic potential cell lines are constructed by this method. The differential expression genes related to tumor metastasis might exist in the human large cell lung cancer cell lines with different metastasis potential.

Identification of genes associated with cold acclimation in blueberry ( Vaccinium corymbosum L.) using a subtractive hybridization approach

Enhanced cold tolerance, including tolerance to winter freezing and spring frosts, is needed for genetic improvement of current highbush blueberry ( Vaccinium corymbosum L.) cultivars. To gain a better understanding of changes in gene expression associated with development of cold tolerance in blueberry and other woody perennials, forward and reverse subtracted cDNA libraries were prepared in such a way to enrich for transcripts that are expressed at higher levels in dormant blueberry flower buds at 400 h and at 0 h of low temperature exposure, respectively. Of the clones picked and single-pass sequenced, 503 clones from the forward subtracted library and 167 clones from the reverse subtracted library had inserts and yielded high quality sequences; and of these, 291 (57.9%) and 51 (31.0%), respectively, were assigned putative identities from BLAST searches of GenBank. From contig analyses to cluster genes of like or identical sequences, 275 unigenes (unique clones) from the forward subtracted library and 99 unigenes from the reverse subtracted library were obtained. Many interesting cold and light-stress related genes were identified from the forward subtracted library and several drought-stress related genes were identified from the reverse subtracted library. Many genes, not previously characterized from blueberry or other woody perennials, encoding putative transcription factors and proteins related to signal transduction were identified from both the forward and reverse subtracted libraries. Eleven genes of interest (six from the forward subtracted library and five from the reverse subtracted library) were selected and their expression was analyzed in floral buds by quantitative real-time RT-PCR over a time course from ∼0 to 1200 h of low temperature exposure. Expression profiles validated the quality of the libraries.

Identifying differentially expressed genes in the dimorphic fungusPenicillium marneffeiby suppression subtractive hybridization

Penicillium marneffei is the only known Penicillium species that exhibits temperature-dependent dimorphic growth. It usually infects immunocompromised individuals and may cause fatal penicilliosis marneffei. Its pathogenicity appears to be intimately related to the dimorphic transition from a mycelial phase in the environment to a yeast phase in the human host. To further understand the molecular mechanisms in the switching process of Penicillium marneffei, differential gene expression analysis was carried out between the mycelial phase and the yeast phase by suppression subtractive hybridization. Five hundred genes from forward and reverse-subtracted cDNA libraries were screened by cDNA array dot blotting and 43 genes were identified to be differentially expressed. These genes were found to be homologs of genes involved in various cellular processes including cell wall synthesis, signal transduction, cell cycle, substance transport, general metabolism and stress response, etc. Six genes were further analyzed using relative quantification real-time reverse transcriptase PCR. It is believed that these genes determine some of the major characteristics of dimorphism in Penicillium marneffei.

A genomic approach to reveal novel genes associated with myotube formation in the model teleost,Takifugu rubripes

Little is known about the transcriptional networks that regulate myotube production in vertebrates. In the present study, we have used a genomic approach to discover novel genes associated with myotube formation in fast muscle of the tiger puffer fish, Takifugu rubripes. The number of fast muscle fibers per myotome increased until 1.2 kg body mass, and subsequent growth was by fiber hypertrophy alone. Forward and reverse subtracted cDNA libraries were prepared from a 180-g (myotube +) and a 3.4-kg (myotube -) fish, and 1,452 expressed sequence tags (ESTs) were obtained. After these ESTs were grouped into nonredundant clusters and housekeeping and structural genes were eliminated, 57 genes were selected and quantitative PCR was used to investigate their expression levels in different tissues from independent groups of myotube(-) and myotube(+) fish acclimated to the same environmental conditions and diet. Eleven novel genes were found to be consistently differentially expressed, but only four showed appropriate tissue-specific expression. These four genes were upregulated 5-25 times in fast muscle of myotube(-) relative to myotube(+) growth stages, while their expression remained unchanged in the other tissues studied. The novel genes identified, which are also present in other vertebrate genomes, may play a role in inhibiting myotube formation in vertebrate muscle.