Serial analysis of gene expression (SAGE) in Plasmodium falciparum: application of the technique to A–T rich genomes

Munasinghe, Anusha; Patankar, Swati; Cook, Brian P.; Madden, Steve L.; Martin, Rodger K.; Kyle, Dennis E.; Shoaibi, Azadeh; Cummings, Leda M.; Wirth, Dyann F.

doi:10.1016/s0166-6851(00)00378-9

Cited by 42 publications

(28 citation statements)

References 30 publications

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“…For example, it is now possible to identify genes that are transcribed in different stages of the parasite's development and also genes that are induced or repressed in response to various stimuli such as immune or drug pressure. For this reason, whole genome expression analyses with the use of high-density microarrays (Hayward et al, 2000) and serial analysis of gene expression (SAGE) (Munasinghe et al, 2000) have been developed for P. falciparum. These new approaches will complement each other to generate data for the Plasmodium research community.…”

Section: Introductionmentioning

confidence: 99%

“…The SAGE technology samples short sequence tags (14 bases) from mRNA transcripts in the population of interest. These tags contain sufficient sequence information to identify, by basic local alignment search tool (BLAST) analysis, the transcript from which each tag was derived (Munasinghe et al, 2000). The frequency of each tag in the SAGE library is an accurate estimate of the abundance of its corresponding mRNA transcript.…”

Section: Introductionmentioning

confidence: 99%

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Serial Analysis of Gene Expression in Plasmodium falciparum Reveals the Global Expression Profile of Erythrocytic Stages and the Presence of Anti-Sense Transcripts in the Malarial Parasite

Patankar

Munasinghe

Shoaibi³

et al. 2001

MBoC

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138

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Serial analysis of gene expression (SAGE) was applied to the malarial parasite Plasmodium falciparum to characterize the comprehensive transcriptional profile of erythrocytic stages. A SAGE library of ϳ8335 tags representing 4866 different genes was generated from 3D7 strain parasites. Basic local alignment search tool analysis of high abundance SAGE tags revealed that a majority (88%) corresponded to 3D7 sequence, and despite the low complexity of the genome, 70% of these highly abundant tags matched unique loci. Characterization of these suggested the major metabolic pathways that are used by the organism under normal culture conditions. Furthermore several tags expressed at high abundance (30% of tags matching to unique loci of the 3D7 genome) were derived from previously uncharacterized open reading frames, demonstrating the use of SAGE in genome annotation. The open platform "profiling" nature of SAGE also lead to the important discovery of a novel transcriptional phenomenon in the malarial pathogen: a significant number of highly abundant tags that were derived from annotated genes (17%) corresponded to antisense transcripts. These SAGE data were validated by two independent means, strand specific reverse transcription-polymerase chain reaction and Northern analysis, where antisense messages were detected in both asexual and sexual stages. This finding has implications for transcriptional regulation of Plasmodium gene expression.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Serial Analysis of Gene Expression in Plasmodium falciparum Reveals the Global Expression Profile of Erythrocytic Stages and the Presence of Anti-Sense Transcripts in the Malarial Parasite

Patankar

Munasinghe

Shoaibi³

et al. 2001

MBoC

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138

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“…Total RNA was extracted from rice leaves 24 h after inoculation with M. grisea by an SDS-phenol extraction protocol (Datson et al, 1999;Munasinghe et al, 2001;Yamamoto et al, 2001). mRNAs containing polyA were isolated using Oligotex-dT column (Qiagen Inc.) as recommended by the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

Gene Expression Profiling in Rice Infected with Rice Blast Fungus using SAGE

Kim¹,

Kim²,

Kim³

et al. 2008

The Plant Pathology Journal

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Rice blast disease, caused by the pathogenic fungus Magnaporthe grisea, is a serious issue in rice (Oryza sativa L.) growing regions of the world. Transcript profiling in rice inoculated with the fungus has been investigated using the transcriptomics technology, serial analysis of gene expression (SAGE). Short sequence tags containing sufficient information which are ten base-pairs representing the unique transcripts were identified by SAGE technology. We identified a total of 910 tag sequences via the GenBank database, and the resulting genes were shown to be up-regulated in all functional categories under the fungal biotic stress. Compared to the compatible interaction, the stress and defense genes in the incompatible interaction appear to be more up-regulated. Particularly, thaumatin-like gene (TLP) was investigated in determining the gene and protein expression level utilizing Northern and Western blotting analyses, resulting in an increase in both the gene and the protein expression level which arose earlier in the incompatible interaction than in the compatible interaction.Keywords : abiotic stress, Magnaporthe grisea, Oryza sativa, pathogenesis-related gene, rice blast, SAGE One of the most devastating diseases in rice, called rice blast, is caused by Magnaporthe grisea (Hamer and Talbot, 1998;Kim et al., 2001). The rice blast disease is a very serious and recurrent issue in rice-growing regions of the world (Talbot, 2003). The infection usually occurs on rice plant leaves where the fungal spores of M. grisea land and attach themselves using the fungal unique adhesive structure called an appressorium (Hamer et al., 1988) to penetrate rice leaf surface by building high turgor pressure (De Jong et al., 1997). Every year, the rice blast infection has the potential to affect the amount of harvested rice by which about sixty million people can be fed (Zeifler et al., 1994), generating significant economical and humanitarian issues in rice-growing areas. It has also been reported that some strains of the fungal M. grisea not only kill rice, but also attack other major cereals and grasses (Valent and Chumley, 1991).In an effort to remedy this agricultural crisis, finding the genes that are able to defend against the fungal biotic stress would be of great value. It is an obvious interest to determine which genes would be involved in the regulation and with what gene expression level in response to M. grisea infection. Transcriptomics can be used to determine the gene expression level of the messenger RNA (mRNA) in a given cell population. Transcriptomics is the study of the transcriptome, which is the set of all mRNA or transcripts produced in a population of cells. The transcriptome varies under stress conditions because all mRNA transcripts in a cell are a reflection of the genes that are being actively expressed under any stress conditions. Two technologies can be useful tools for transcriptomics. One of the technologies is microarray analysis, which utilizes labeled cDNAs hybridized to an array of DNA element...

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“…One microgram of total RNA was reverse transcribed in a 20-l reaction mixture containing 0.5 M oligo(dT)-Heel primer (5Ј-CCAGTG TCTTGAGCAGTGACT 18 VN-3Ј, where V is A, C, or G and N is any nucleotide), 1ϫ SuperScript buffer (Invitrogen, Carlsbad, Calif.), 10 mM dithiothreitol, 0.5 mM each deoxynucleoside triphosphate (dNTP), and 200 U of SuperScript II reverse transcriptase (Invitrogen). The reaction was allowed to proceed for 50 min at 42°C.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, the serial and parallel analysis of sequence tags increases data output by several orders of magnitude, generating libraries that are both quantitative and comprehensive. Although SAGE was developed initially for medical research, it has been successfully extended to the analysis of gene expression in a diverse number of species, such as the yeast Saccharomyces cerevisiae (28), rice seedlings (17), and the malarial parasite Plasmodium falciparum (18).…”

mentioning

confidence: 99%

Modified Serial Analysis of Gene Expression Method for Construction of Gene Expression Profiles of Microbial Eukaryotic Species

Coyne

Burkholder

Feldman³

et al. 2004

Appl Environ Microbiol

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Serial analysis of gene expression (SAGE) is a powerful approach for the identification of differentially expressed genes, providing comprehensive and quantitative gene expression profiles in the form of short tag sequences. Each tag represents a unique transcript, and the relative frequencies of tags in the SAGE library are equal to the relative proportions of the transcripts they represent. One of the major obstacles in the preparation of SAGE libraries from microorganisms is the requirement for large amounts of starting material (i.e., mRNA). Here, we present a novel approach for the construction of SAGE libraries from small quantities of total RNA by using Y linkers to selectively amplify 3 cDNA fragments. To validate this method, we constructed comprehensive gene expression profiles of the toxic dinoflagellate Pfiesteria shumwayae. SAGE libraries were constructed from an actively toxic fish-fed culture of P. shumwayae and from a recently toxic alga-fed culture. P. shumwayae-specific gene transcripts were identified by comparison of tag sequences in the two libraries. Representative tags with frequencies ranging from 0.026 to 3.3% of the total number of tags in the libraries were chosen for further analysis. Expression of each transcript was confirmed in separate control cultures of toxic P. shumwayae. The modified SAGE method described here produces gene expression profiles that appear to be both comprehensive and quantitative, and it is directly applicable to the study of gene expression in other environmentally relevant microbial species.Serial analysis of gene expression (SAGE) is a powerful and efficient method for compiling quantitative gene expression profiles of specific tissue or cell types (26-28). In contrast to conventional cDNA libraries, this technique allows the simultaneous analysis of gene expression for thousands of transcripts by cataloging short (10-base) diagnostic sequence tags located at a specific site near the 3Ј end of each gene transcript. The SAGE method has several advantages over other methods for analysis of differential gene expression (see, for example, references 6, 12, and 13). No prior knowledge of a gene sequence is required, and the sequence data generated can be archived for comparison to future libraries. In addition, the serial and parallel analysis of sequence tags increases data output by several orders of magnitude, generating libraries that are both quantitative and comprehensive. Although SAGE was developed initially for medical research, it has been successfully extended to the analysis of gene expression in a diverse number of species, such as the yeast Saccharomyces cerevisiae (28), rice seedlings (17), and the malarial parasite Plasmodium falciparum (18).In spite of the value of SAGE libraries, their construction and analysis can be challenging. A major drawback to the original SAGE protocol is the initial requirement for microgram quantities of mRNA. For biological materials available only in limited quantities, PCR-based methods that allow construction of S...

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Serial analysis of gene expression (SAGE) in Plasmodium falciparum: application of the technique to A–T rich genomes

Cited by 42 publications

References 30 publications

Serial Analysis of Gene Expression in Plasmodium falciparum Reveals the Global Expression Profile of Erythrocytic Stages and the Presence of Anti-Sense Transcripts in the Malarial Parasite

Serial Analysis of Gene Expression in Plasmodium falciparum Reveals the Global Expression Profile of Erythrocytic Stages and the Presence of Anti-Sense Transcripts in the Malarial Parasite

Gene Expression Profiling in Rice Infected with Rice Blast Fungus using SAGE

Modified Serial Analysis of Gene Expression Method for Construction of Gene Expression Profiles of Microbial Eukaryotic Species

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