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.     

Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu.     

A metagenomic approach to the identification of UDP-glucose 4-epimerase as a menadione resistance protein

Quinones are potentially toxic agents that generate reactive oxygen species (ROS) upon reduction. We screened a metagenomic library for a menadione resistance gene to identify UDP-glucose 4-epimerase (UGE). Escherichia coli carrying the gene became resistant to various quinones, but not to ROS. Because UGE is involved in the biosynthesis of lipopolysaccharides, it may have contributed to forming a permeability barrier against lipophilic quinones.     

Identification of differentially expressed genes in Tetrahymena thermophila in response to dichlorodiphenyltrichloroethane (DDT) by suppression subtractive hybridization

The insecticide dichlorodiphenyltrichloroethane (DDT) is persistent in the environment, and continues to cause health problems. Tetrahymena has potential as a model organism for assaying low levels of DDT and for analysing the mechanisms of its toxicity. We constructed the suppression subtractive hybridization library of T. thermophila exposed to DDT, and screened out 90 Expressed Sequence Tags whose expressions were significantly up- or downregulated with DDT treatment. From this, a series of important genes related to the DDT metabolism and detoxification were discovered, such as P450 gene, glutathione S-transferase gene and sterol carrier protein 2 gene. Furthermore, their expressions under different concentrations of DDT treatment were detected by real-time fluorescent quantitative PCR. The results show that Tetrahymena is a relevant and useful model organism for detecting DDT in the environment and for discovering biomarkers that can be used to develop specific bio-reporters at the molecular and genomic levels.     

Identification of differentially expressed genes in 4-day axolotl limb blastema by suppression subtractive hybridization

The goal of our study was the identification of up-regulated genes during axolotl (Ambystoma mexicanum) hindlimb regeneration 4 days after amputation using suppression subtractive hybridization (SSH). Approximately 400 clones that harbored upregulated genes in regenerating blastema tissue were selected for sequence analysis. A BLAST homology search against NCBI non-redundant database and an ambystoma EST database revealed 102 clones that showed homology to known sequences in GenBank with annotated function, 31 were known genes without known function, 74 were novel and 72 belonged to mitochondrial sequences. Differential expression of Hmox1, Orc4L, Pls3, Fen-1, Mcm7 and Mmp3/10a was confirmed using qRT-PCR analysis. Among all genes, only Mmp3/10a has been previously described as involved in limb regeneration. Other important identified genes belong to the group of cell cycle regulators (Orc4L, Nasp, Skp1A and Mcm7, the latter being a possible proliferative marker), those involved in protein synthesis and transport (Sec63, Srp72, Sara2) and V- ATPase pump. The novel genes we identified might be important for the process of blastema formation and the onset of cell proliferation in a regenerating limb.     

柚树(Citrus grandis)叶片光合作用对补增UV-B辐射的响应

生长在人工光照 4 0 0μmol m- 2 s- 1 下的柚树幼树光合速率的最大值为 1 0 .2± 0 .5μmol m- 2 s- 1 ;而补增UV-B辐射 ( 3.8-4 .2μW cm- 2 ,2 4 5～ 2 97nm,4 5d)的叶片则为 6.4± 0 .8μmol m- 2 s- 1 ,较对照植株降低37.2 %。对照植物的表观量子产率 (固定 mol CO2 mol- 1量子 )为 0 .0 75± 0 .0 1 2 ,而经 UV-B辐射处理植株则为0 .0 4 1± 0 .0 0 8,明显较对照植株低。UV-B辐射处理使植株叶片的光呼吸和不包括光呼吸的 CO2 补偿点增高。对照植株叶片的最大值的 CO2 羧化速率 (μmol m- 2 s- 1 )为 57.1± 1 .5μmol m- 2 s- 1 ,较 UV-B辐射处理的高30 .9% ,而 UV-B辐射处理的植株的光合电子传递速率较对照低 30 %。同时 UV-B辐射植株叶片有较低的光能转化效率 ,其较对照低 39.1 % ,叶片亦含有较低的叶绿素含量。结果表明 ,UV-B辐射明显抑制叶片光合羧化速率和光合电子传递速率 ,UV-B辐射可能抑制包括 Rubisco羧化作用在内的多个光合生理过程 ,降低叶片光合速率。柚树叶片对 UV-B辐射敏感 ,选育抗 UV-B辐射的柚树品种势在必行。     

Identification of early salt stress response genes in tomato root by suppression subtractive hybridization and microarray analysis

High salinity is one of the most serious threats to crop production. To understand the molecular basis of plant responses to salt stress better, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes involved in the early stage of tomato responses to severe salt stress. First, SSH libraries were constructed for the root tissue of two cultivated tomato (Solanum lycopersicum) genotypes: LA2711, a salt-tolerant cultivar, and ZS-5, a salt-sensitive cultivar, to compare salt treatment and non-treatment plants. Then a subset of clones from these SSH libraries were used to construct a tomato cDNA array and microarray analysis was carried out to verify the expression changes of this set of clones upon a high concentration of salt treatment at various time points compared to the corresponding non-treatment controls. A total of 201 non-redundant genes that were differentially expressed upon 30 min of severe salt stress either in LA2711 or ZS-5 were identified from microarray analysis; most of these genes have not previously been reported to be associated with salt stress. The diversity of the putative functions of these genes indicated that salt stress resulted in a complex response in tomato plants.     

Suppression subtractive hybridization analysis reveals expression of conserved and novel genes in male accessory glands of the ant Leptothorax gredleri

An issue arising from recent progress in establishing the placental mammal Tree of Life concerns the nomenclature of high-level clades. Fortunately, there are now several well-supported clades among extant mammals that require unambiguous, stable names. Although the International Code of Zoological Nomenclature does not apply above the Linnean rank of family, and while consensus on the adoption of competing systems of nomenclature does not yet exist, there is a clear, historical basis upon which to arbitrate among competing names for high-level mammalian clades. Here, we recommend application of the principles of priority and stability, as laid down by G.G. Simpson in 1945, to discriminate among proposed names for high-level taxa. We apply these principles to specific cases among placental mammals with broad relevance for taxonomy, and close with particular emphasis on the Afrotherian family Tenrecidae. We conclude that no matter how reconstructions of the Tree of Life change in years to come, systematists should apply new names reluctantly, deferring to those already published and maximizing consistency with existing nomenclature.     

Suppression subtractive hybridization library prepared from the copepod Calanus finmarchicus exposed to a sublethal mixture of environmental stressors

A library of expressed sequence tags (ESTs) was constructed by the use of suppression subtractive hybridization polymerase chain reaction (SSH PCR) technique from the marine copepod Calanus finmarchicus. Samples used were from controls (seawater, 10 °C) and exposed (sublethal mixture) individuals. The sublethal exposure regime consisted of a mixture of mono ethanol amine (MEA), water-soluble fractions of oil (WSFs), copper (Cu) and elevated temperature (17 °C). The resulting 189 unique ESTs consisted of 127 putatively up-regulated genes and 54 putatively down-regulated genes. Annotation analyses revealed altered expression of a wide variety of genes, among these putative heat shock protein 90 (HSP-90), antioxidants (thioredoxin reductase, glutathione peroxidase) and cytochrome P450 enzymes. In addition, sequences showing high similarity to enzymes involved in fatty acid metabolism, energy metabolism and amine handling were found further confirming the effects of the exposure. The annotated sequences are discussed in relation to the present exposure as well as known physiological mechanisms known in C. finmarchicus and related copepod species. The sequenced ESTs from our C. finmarchicus library will provide an excellent tool for future studies on this species, both from a toxicogenomic and systems biology point of view.     

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp.