Construction and analysis of a SSH cDNA library of Eucalyptus grandis × Eucalyptus urophylla 9224 induced by Cylindrocladium quinqueseptatum

Leaf blight caused by Cylindrocladium quinqueseptatum Boedijn & Reitsma is a major disease occurring in Eucalyptus plantlets and saplings worldwide. Current studies on the resistance to this disease in eucalypti have focused primarily on analyzing the relationship between the disease and the metabolic products from the anatomic structure of Eucalyptus leaves and their physiological responses to pathogen invasion. There have been no reports on the mechanisms of molecular responses. Previous research suggests that Eucalyptus grandis × Eucalyptus urophylla 9224 is the highest resistant cultivar among the 11 ones planted in Fujian Province. Therefore, in this study, the molecular responses of E. grandis × E. urophylla 9224 to C. quinqueseptatum were investigated by means of suppression subtractive hybridization (SSH). In total, 928 clones were obtained from the forward SSH cDNA library of E. grandis × E. urophylla 9224, and 25 up-regulated genes were identified by screening the library using the method of reverse northern hybridization. The results show that among these, 23 up-regulated genes with known functions were involved in signal transduction, plant resistance response, photosynthesis, energy transfer, protein metabolism, and plant growth.

Comparative transcriptome analysis of yeast strains carrying slt2, rlm1, and pop2 deletions

The function of the genes SLT2 (encoding the Mpk1 protein), RLM1, and POP2 have previously been related to several stress responses in yeasts. DNA arrays have been used to identify differences among the transcriptomes of a Saccharomyces cerevisiae wild type strain and its derivative Δslt2, Δrlm1, and Δpop2 mutants. Correspondence analyses indicate that the vast majority of genes that show lower expression in Δrlm1 also show lower expression in Δslt2. In contrast, there is little overlap between the results of the transcriptome analyses of the Δpop2 strain and the Δslt2 or Δrlm1 strains. The DNA array data were validated by reverse Northern blotting and chromatin immunoprecipitation (ChIp). ChIp assays demonstrate Rlm1p binding to specific regions of the promoters of two genes that show expression differences between the Δrlm1 mutant and wild type strains. Interestingly, RLM1 deletion decreases the transcription of SLT2, encoding the Mpk1p kinase that phosphorylates Rlm1p, suggesting a feedback control in the signal transduction pathway. Also, deletion of RLM1 causes a decrease in the mRNA level of KDX1, which is paralogous to SLT2. In contrast, deletion of POP2 is accompanied by an increase of both SLT2 and KDX1 levels. We show that SLT2 mRNA increase in the Δpop2 strain is due to a decrease in RNA turnover, consistent with the expected loss of RNA-deadenylase activity in this strain.

Identification of stress-induced genes from the drought-tolerant plant Prosopis juliflora (Swartz) DC. through analysis of expressed sequence tags

Abiotic stresses such as cold, salinity, drought, wounding, and heavy metal contamination adversely affect crop productivity throughout the world. Prosopis juliflora is a phreatophyte that can tolerate severe adverse environmental conditions such as drought, salinity, and heavy metal contamination. As a first step towards the characterization of genes that contribute to combating abiotic stress, construction and analysis of a cDNA library of P. juliflora genes is reported here. Random expressed sequence tag (EST) sequencing of 1750 clones produced 1467 high-quality reads. These clones were classified into functional categories, and BLAST comparisons revealed that 114 clones were homologous to genes implicated in stress response(s) and included heat shock proteins, metallothioneins, lipid transfer proteins, and late embryogenesis abundant proteins. Of the ESTs analyzed, 26% showed homology to previously uncharacterized genes in the databases. Fifty-two clones from this category were selected for reverse Northern analysis: 21 were shown to be upregulated and 16 downregulated. The results obtained by reverse Northern analysis were confirmed by Northern analysis. Clustering of the 1467 ESTs produced a total of 295 contigs encompassing 790 ESTs, resulting in a 54.2% redundancy. Two of the abundant genes coding for a nonspecific lipid transfer protein and late embryogenesis abundant protein were sequenced completely. Northern analysis (after polyethylene glycol stress) of the 2 genes was carried out. The implications of the analyzed genes in abiotic stress tolerance are also discussed.

Identification of Genes Regulated by Low Temperature in Pachysandra terminalis Sieb. et Zucc Using cDNA Differential Display

Complementary Deoxyribonucleic Acid (cDNA) differential display and reverse Northern dot blot were used to identify genes in Pachysandra terminalis Sieb. & Zucc., a cold-tolerant plant, that are regulated by low temperatures. Rooted cuttings were obtained from stock plants that had been maintained in a greenhouse at 24 °C. These cuttings were subjected to the following cold treatments: 2 weeks at 12 °C, 48 hours at 4 °C, 48 hours at 0 °C, and 4 hours at –1 °C. Following leaf tissue analysis of treated and control plants, some stress-related genes and many novel genes were identified. Northern blot hybridization demonstrated that all novel genes were regulated by the cold treatments.

Identification of Differentially Expressed Genes in Metastatic and Non-Metastatic Nasopharyngeal Carcinoma Cells by Suppression Subtractive Hybridization

Background & Objective: Nasopharyngeal carcinoma (NPC) is an epithelial neoplasm with high occurrence rates in southern China. The disease often metastasizes to regional lymphnodes at a very early stage. Local recurrences and metastasis occur frequently in patients with NPC and are a leading cause of death, despite improvements on treatment modalities. The molecular mechanism underlying the metastasis of nasopharyngeal carcinoma remains poorly understood, however, and requires additional elucidation. The aim of this study was to explore possible NPC gene candidates that may play key roles in NPC metastasis. Methods: Subtractive suppression hybridization (SSH) was performed to isolate differentially expressed clones between the metastatic 5-8F and non-metastatic 6-10B nasopharyngeal carcinoma cell lines. Differentially expressed clones were screened and confirmed by reverse Northern blotting. The sequences of cDNA fragments were subsequently analyzed and compared to known sequences in Genbank. Results & Discussion: The SSH library contained thousands of positive clones. Random analysis of 300 clones by PCR demonstrated that 269 clones contained inserted fragments. Reverse Northern blot confirmed that 20 out of 192 clones examined were significantly up-regulated in the 5-8F cell line. Among these 20 clones, 16 were previously identified genes (flotilin-2, ezrin, pim-3, fli-1, mel, neugrin, znf216, ASB1, raly, UBE2A, keratin6A, TMED7, EIF3S9, FTL, two ribosomal proteins RPL21 and RPL16), two were predicted genes (c9orf74 and MDS006), and two sequences shared no homology with known genes listed in GenBank and may represent novel genes. The proposed functions of the genes identified in this study include cell signal transduction, cell survival, transcription regulation, cell mobility, protein synthesis, and DNA damage repair. Flotillin-2, fli-1, pim-3 and ezrin have previously been reported to be associated with tumor metastasis and progression. The remaining up-regulated genes identified in this study have not been reported to be markers of metastasis and may represent new candidates of NPC metastasis-related genes. The Results of this study may provide novel points of therapeutic intervention for NPC.

Good quality Vitis RNA obtained from an adapted DNA isolation protocol

<p style="text-align: justify;">Grapevine is a woody plant, whose high carbohydrate and phenolic compound contents usually interferes with nucleic acid isolation. After we tried several protocols for isolating RNA from the <em>Vitis</em> rootstock Richter- 110 (R-110) with little or no success, we adapted a method reported to be satisfactory for grapevine DNA isolation, to extract RNA. With slight protocol modifications, we succeeded to obtain polysaccharide- and phenolic-free RNA preparations from all vegetative tissues, without excessive sample handling. RNA isolated by the reported method permitted to obtain highly pure mRNA (messenger RNA) to construct a cDNA (complementary DNA) library and allowed gene transcription analysis by reverse Northern, which guarantees RNA integrity. This method may also be suitable for other plant species with high polysaccharide or phenolic contents.</p>