Suppressive subtractive hybridization and differential screening identified genes differentially expressed in yeast and mycelial forms ofOphiostoma piceae

The microsporidium Nosema ceranae is a high prevalent parasite of the European honey bee (Apis mellifera). This parasite is spreading across the world into its novel host. The developmental process, and some mechanisms of N. ceranae-infected honey bees, has been studied thoroughly; however, few studies have been carried out in the mechanism of gene expression in N. ceranae during the infection process. We therefore performed the suppressive subtractive hybridization (SSH) approach to investigate the candidate genes of N. ceranae during its infection process. All 96 clones of infected (forward) and non-infected (reverse) library were dipped onto the membrane for hybridization. A total of 112 differentially expressed sequence tags (ESTs) had been sequenced. For the host responses, 20% of ESTs (13 ESTs, 10 genes, and 1 non-coding RNA) from the forward library and 93.6% of ESTs (44 ESTs, 28 genes) from the reverse library were identified as differentially expressed genes (DEGs) of the hosts. A high percentage of DEGs involved in catalytic activity and metabolic processes revealed that the host gene expression change after N. ceranae infection might lead to an unbalance of physiological mechanism. Among the ESTs from the forward library, 75.4% ESTs (49 ESTs belonged to 24 genes) were identified as N. ceranae genes. Out of 24 N. ceranae genes, nine DEGs were subject to real-time quantitative reverse transcription PCR (real-time qRT-PCR) for validation. The results indicated that these genes were highly expressed during N. ceranae infection. Among nine N. ceranae genes, one N. ceranae gene (AAJ76_1600052943) showed the highest expression level after infection. These identified differentially expressed genes from this SSH could provide information about the pathological effects of N. ceranae. Validation of nine up-regulated N. ceranae genes reveal high potential for the detection of early nosemosis in the field and provide insight for further applications.

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Microarray screening of suppression subtractive hybridization-PCR cDNA libraries identifies novel RNAs regulated by dehydration in the rat supraoptic nucleus

Physiological Genomics ◽

10.1152/physiolgenomics.00229.2005 ◽

2006 ◽

Vol 24 (2) ◽

pp. 163-172 ◽

Cited By ~ 17

Author(s):

Mohamed T. Ghorbel ◽

Greig Sharman ◽

Charles Hindmarch ◽

Kevin G. Becker ◽

Tanya Barrett ◽

...

Keyword(s):

General Circulation ◽

Supraoptic Nucleus ◽

Subtractive Hybridization ◽

Biologically Active ◽

Differentially Expressed ◽

Cdna Libraries ◽

Suppressive Subtractive Hybridization ◽

Cdna Clones ◽

Microarray Screening ◽

Mechanistic Basis

The magnocellular neurons (MCNs) of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus are the principal site of biosynthesis of prepropeptide precursor of the antidiuretic hormone vasopressin (VP). This precursor is processed during anterograde axonal transportation to terminals in the posterior pituitary gland, where biologically active VP is stored until release into the general circulation in response to physiological activation of the SON by osmotic cues. By binding to V2-type receptors located in the kidney, VP decreases the amount of water lost in urine. Osmotic activation of the SON is accompanied by a dramatic morphological and functional remodeling. We have sought to understand the mechanistic basis of this plasticity in terms of the differential expression of genes. To identify such genes, we adopted an unbiased global approach based on suppressive subtractive hybridization-polymerase chain reaction (SSH-PCR) Using this method, we generated libraries of clones putatively differentially expressed in control vs. dehydrated SON. To rapidly screen these libraries, 1,152 clones were subjected to microarray analysis, resulting in the identification of 459 differentially expressed transcripts. cDNA clones corresponding to 56 of these RNAs were sequenced, revealing many of them to be novel expressed sequence tags (ESTs). Four transcripts were shown by in situ hybridization (ISH) to be significantly up- or downregulated in the SON after dehydration. These genes may represent novel effectors or mediators of SON physiological remodeling.

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Suppressive Subtractive Hybridization of and Differences in Gene Expression Content of Calcifying and Noncalcifying Cultures of Emiliania huxleyi Strain 1516

Applied and Environmental Microbiology ◽

10.1128/aem.71.5.2564-2575.2005 ◽

2005 ◽

Vol 71 (5) ◽

pp. 2564-2575 ◽

Cited By ~ 23

Author(s):

Binh Nguyen ◽

Robert M. Bowers ◽

Thomas M. Wahlund ◽

Betsy A. Read

Keyword(s):

Time Course ◽

Expression Profiles ◽

Expression Patterns ◽

Differentially Expressed Gene ◽

Subtractive Hybridization ◽

Emiliania Huxleyi ◽

Marine Ecology ◽

Differentially Expressed ◽

Suppressive Subtractive Hybridization ◽

Pcr Analysis

ABSTRACT The marine coccolithophorid Emiliania huxleyi is a cosmopolitan alga intensely studied in relation to global carbon cycling, biogeochemistry, marine ecology, and biomineralization processes. The biomineralization capabilities of coccolithophorids have attracted the attention of scientists interested in exploiting this ability for the development of materials science and biomedical and biotechnological applications. Although it has been well documented that biomineralization in E. huxleyi is promoted by growth under phosphate-limited conditions, the genes and proteins that govern the processes of calcification and coccolithogenesis remain unknown. Suppressive subtractive hybridization (SSH) libraries were constructed from cultures grown in phosphate-limited and phosphate-replete media as tester and driver populations for reciprocal SSH procedures. Positive clones from each of the two libraries were randomly selected, and dot blotting was performed for the analysis of expression patterns. A total of 513 clones from the phosphate-replete library and 423 clones from the phosphate-limited library were sequenced, assembled, and compared to sequences in GenBank using BLASTX. Of the 103 differentially expressed gene fragments from the phosphate-replete library, 34% showed significant homology to other known proteins, while only 23% of the 65 differentially expressed gene fragments from the phosphate-limited library showed homology to other proteins. To further assess mRNA expression, real-time RT-PCR analysis was employed and expression profiles were generated over a 14-day time course for three clones from the phosphate-replete library and five clones from the phosphate-limited library. The fragments isolated provide the basis for future cloning of full-length genes and functional analysis.

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Identification of novel differentially expressed zooxanthellal genes from Aiptasia-Symbiodinium endosymbiosis through SDS-based RNA purification

10.1101/2020.09.01.277251 ◽

2020 ◽

Author(s):

Bo-Nien Chen ◽

Paching Song ◽

Ming-Chyuan Chen ◽

Ming-Chang Hong

Keyword(s):

Starch Synthesis ◽

Subtractive Hybridization ◽

Differentially Expressed ◽

Suppressive Subtractive Hybridization ◽

Functional Genes ◽

Cellular Processes ◽

Physiological Processes ◽

Rna Purification ◽

Next Generation Sequencing Ngs ◽

Cycle Regulation

AbstractEndosymbiosis between dinoflagellates and cnidarian hosts first occurred more than 200 million years ago; however, symbiosis-specific genes and cellular processes involved in the establishment, maintenance, and breakdown of endosymbiosis remain unclear. Therefore, this study aimed to identify the zooxanthellal genes associated with the aforementioned biological processes during endosymbiosis in Aiptasia-Symbiodinium endosymbionts. Here, zooxanthellae isolates were treated with 0.02% SDS to decrease potential host RNA contamination and to enhance the identification of novel symbiosis/nonsymbiosis-associated differentially expressed zooxanthellal genes through suppressive subtractive hybridization (SSH) and next-generation sequencing (NGS) methods. Consequently, among 214 symbiosis-specific transcripts identified herein that displayed identity to only 5.6% of host-derived transcripts, 64% were well-known functional genes. In the nonsymbiotic stage, 181 differentially expressed transcripts were identified, of which 64.1% belonged to well-known functional genes. BLAST revealed that 8 categories of cellular processes were significantly induced in symbiotic or nonsymbiotic zooxanthellae. Together with the results of quantitative analysis, the results revealed that photosynthesis, flagellate biosynthesis and motility, stress-induced responses, cell wall biosynthesis, starch synthesis and transport, lipid biosynthesis and metabolism, host/symbiont immune response, intercellular communication, cell growth, and cell cycle regulation were the major cellular processes occurring in symbiotic/nonsymbiotic stages. The present results provide insights into the mechanisms involved in regulating the different physiological processes in symbiotic/nonsymbiotic zooxanthellae and may guide future studies.

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