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Microarray analyses of gene expression during the Tetrahymena thermophila life cycle.

Miao W, Xiong J, Bowen J, Wang W, Liu Y, Braguinets O, Grigull J, Pearlman RE, Orias E, Gorovsky MA - PLoS ONE (2009)

Bottom Line: Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation.Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions.New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Rochester, Rochester, New York, USA. miaowei530@yeah.net

ABSTRACT

Background: The model eukaryote, Tetrahymena thermophila, is the first ciliated protozoan whose genome has been sequenced, enabling genome-wide analysis of gene expression.

Methodology/principal findings: A genome-wide microarray platform containing the predicted coding sequences (putative genes) for T. thermophila is described, validated and used to study gene expression during the three major stages of the organism's life cycle: growth, starvation and conjugation.

Conclusions/significance: Of the approximately 27,000 predicted open reading frames, transcripts homologous to only approximately 5900 are not detectable in any of these life cycle stages, indicating that this single-celled organism does indeed contain a large number of functional genes. Transcripts from over 5000 predicted genes are expressed at levels >5x corrected background and 95 genes are expressed at >250x corrected background in all stages. Transcripts homologous to 91 predicted genes are specifically expressed and 155 more are highly up-regulated in growing cells, while 90 are specifically expressed and 616 are up-regulated during starvation. Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation. The patterns of gene expression during conjugation correlate well with the developmental stages of meiosis, nuclear differentiation and DNA elimination. The relationship between gene expression and chromosome fragmentation is analyzed. Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions. New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions.

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Background determination and normalization controls.(A) Background signal intensities during growth (L-l to L-h), starvation (S-0 to S-24) and conjugation (C-0 to C-18) stages were determined using 4308 different random probes on each array. The signal intensities shown for these probes are the average of 12924 values for triplicate growth and starvation samples and 8616 values for duplicate conjugation samples. The bars represent the standard errors. (B, C) Relative levels of HHT3 and SerH3 mRNAs at all 20 stages. The results shown here are the average of triplicate growth and starvation samples and duplicate conjugation samples, and the bars represent the standard errors.
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pone-0004429-g001: Background determination and normalization controls.(A) Background signal intensities during growth (L-l to L-h), starvation (S-0 to S-24) and conjugation (C-0 to C-18) stages were determined using 4308 different random probes on each array. The signal intensities shown for these probes are the average of 12924 values for triplicate growth and starvation samples and 8616 values for duplicate conjugation samples. The bars represent the standard errors. (B, C) Relative levels of HHT3 and SerH3 mRNAs at all 20 stages. The results shown here are the average of triplicate growth and starvation samples and duplicate conjugation samples, and the bars represent the standard errors.

Mentions: We wished to establish that the microarray platform described here produced reliable results that correlated with global measurements of gene expression and analyses of expression of specific genes performed in Tetrahymena by other methods. To this end, we established background levels of hybridization (negative controls) and demonstrated, at the level of individual genes, that the expression values obtained were reproducible among biological replicates. To estimate non-specific, background binding, included on each array were 4308 randomly generated oligonucleotide probes comparable in length and GC content to the experimental probes on the array. The low, average mean signal for these negative probes, 33 arbitrary units (AU; Figure 1A), represents the best estimate of methodological background–due to preparation of samples, array manufacture and processing–but does not indicate to what extent probes on the array might cross-hybridize weakly to the labeled RNA sequences. We estimated these weak cross-hybridizations (as described under Materials and Methods, using the approach described in [38]) by plotting the distribution of signal intensities at different levels of background subtraction (see Figure 2 for the 2 hr conjugation sample). As was also observed by Wei [38] for sea urchin microarrays, at 3× subtraction the distribution in the Tetrahymena microarray has converged to a robust profile that retains no hint of the very large non-specific hybridization peak observed in the unsubtracted distribution. Thus 3× subtraction of the negative control background seems sufficient to remove the vast majority (if not all) of the methodological and non-specific cross-hybridization background. Wei et al [38] were able to conclude from independent data available in sea urchins (similar data are not available in Tetrahymena) that this 3× subtraction was not likely to eliminate signals from known transcripts of very low abundance. Based on all these findings, we define 3× the methodological background (99 AUs) as “1× corrected background” in subsequent analyses.


Microarray analyses of gene expression during the Tetrahymena thermophila life cycle.

Miao W, Xiong J, Bowen J, Wang W, Liu Y, Braguinets O, Grigull J, Pearlman RE, Orias E, Gorovsky MA - PLoS ONE (2009)

Background determination and normalization controls.(A) Background signal intensities during growth (L-l to L-h), starvation (S-0 to S-24) and conjugation (C-0 to C-18) stages were determined using 4308 different random probes on each array. The signal intensities shown for these probes are the average of 12924 values for triplicate growth and starvation samples and 8616 values for duplicate conjugation samples. The bars represent the standard errors. (B, C) Relative levels of HHT3 and SerH3 mRNAs at all 20 stages. The results shown here are the average of triplicate growth and starvation samples and duplicate conjugation samples, and the bars represent the standard errors.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2636879&req=5

pone-0004429-g001: Background determination and normalization controls.(A) Background signal intensities during growth (L-l to L-h), starvation (S-0 to S-24) and conjugation (C-0 to C-18) stages were determined using 4308 different random probes on each array. The signal intensities shown for these probes are the average of 12924 values for triplicate growth and starvation samples and 8616 values for duplicate conjugation samples. The bars represent the standard errors. (B, C) Relative levels of HHT3 and SerH3 mRNAs at all 20 stages. The results shown here are the average of triplicate growth and starvation samples and duplicate conjugation samples, and the bars represent the standard errors.
Mentions: We wished to establish that the microarray platform described here produced reliable results that correlated with global measurements of gene expression and analyses of expression of specific genes performed in Tetrahymena by other methods. To this end, we established background levels of hybridization (negative controls) and demonstrated, at the level of individual genes, that the expression values obtained were reproducible among biological replicates. To estimate non-specific, background binding, included on each array were 4308 randomly generated oligonucleotide probes comparable in length and GC content to the experimental probes on the array. The low, average mean signal for these negative probes, 33 arbitrary units (AU; Figure 1A), represents the best estimate of methodological background–due to preparation of samples, array manufacture and processing–but does not indicate to what extent probes on the array might cross-hybridize weakly to the labeled RNA sequences. We estimated these weak cross-hybridizations (as described under Materials and Methods, using the approach described in [38]) by plotting the distribution of signal intensities at different levels of background subtraction (see Figure 2 for the 2 hr conjugation sample). As was also observed by Wei [38] for sea urchin microarrays, at 3× subtraction the distribution in the Tetrahymena microarray has converged to a robust profile that retains no hint of the very large non-specific hybridization peak observed in the unsubtracted distribution. Thus 3× subtraction of the negative control background seems sufficient to remove the vast majority (if not all) of the methodological and non-specific cross-hybridization background. Wei et al [38] were able to conclude from independent data available in sea urchins (similar data are not available in Tetrahymena) that this 3× subtraction was not likely to eliminate signals from known transcripts of very low abundance. Based on all these findings, we define 3× the methodological background (99 AUs) as “1× corrected background” in subsequent analyses.

Bottom Line: Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation.Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions.New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Rochester, Rochester, New York, USA. miaowei530@yeah.net

ABSTRACT

Background: The model eukaryote, Tetrahymena thermophila, is the first ciliated protozoan whose genome has been sequenced, enabling genome-wide analysis of gene expression.

Methodology/principal findings: A genome-wide microarray platform containing the predicted coding sequences (putative genes) for T. thermophila is described, validated and used to study gene expression during the three major stages of the organism's life cycle: growth, starvation and conjugation.

Conclusions/significance: Of the approximately 27,000 predicted open reading frames, transcripts homologous to only approximately 5900 are not detectable in any of these life cycle stages, indicating that this single-celled organism does indeed contain a large number of functional genes. Transcripts from over 5000 predicted genes are expressed at levels >5x corrected background and 95 genes are expressed at >250x corrected background in all stages. Transcripts homologous to 91 predicted genes are specifically expressed and 155 more are highly up-regulated in growing cells, while 90 are specifically expressed and 616 are up-regulated during starvation. Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation. The patterns of gene expression during conjugation correlate well with the developmental stages of meiosis, nuclear differentiation and DNA elimination. The relationship between gene expression and chromosome fragmentation is analyzed. Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions. New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions.

Show MeSH
Related in: MedlinePlus