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Exploring the potential of a Functional Gene Array (FGA) for fluvial biofilms

ABSTRACT: Microarrays offer a comprehensive tool for a system-wide analysis of cell functioning based on the simultaneous detection of the activity of thousands of genes. While transcriptome analyses are usually related with the study of gene expression and regulation within single species in laboratory experiments or environmental samples, in the present study, the possibility of studying gene expression in river biofilm communities was explored. To this aim a Functional Gene array (FGA), based on consensus sequences from genes of key physiological processes, was designed including 83 functional genes chosen to reflect several essential biochemical pathways and specific stress response pathways. Probes of these genes were designed from consensus sequences from up to 6 microalgal species (diatoms and chlorophytes). Furthermore, species specific probes were included resulting in 1556 unique oligonucleotide probes for 83 different genes. RNA extracted from Chlamydomonas reinhardtii, Scenedesmus vacuolatus and multi-species biofilms was then hybridized to oligonucleotide arrays at different hybridization temperatures (55, 60 and 65ºC). Signal intensity was affected by sequence divergence but results showed that hybridisation temperature of 55ºC allowed a good compromise between cross-hybridization and specificity. Due to the limited number of probes and available sequence information the designed FGAs is at present particularly useful for the comparison of similar biofilms exposed to different conditions in laboratory experimental studies.

ORGANISM(S): Chlamydomonas Bacillariophyta Scenedesmus vacuolatus Chlorophyta environmental samples Chlamydomonas reinhardtii Tetradesmus obliquus

PROVIDER: GSE22496 | GEO | 2012/12/31

SECONDARY ACCESSION(S): PRJNA128659

REPOSITORIES: GEO

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Project description:The protozoan Ichthyophthirius multifiliis (Ich) is a eukaryotic ciliate parasite of freshwater fish. Ich causes ichthyophthiriosis or ‘white spot disease’ characterized by white cysts covering the host skin and gills. The parasite is responsible for high mortalities and severe economic losses to farmed species as well as to ornamental species of fish. Despite the global importance of Ich, little is known about the genetic processes underlying its development and infectivity. Ich has three main life-stages, an infective theront, a parasitic trophont, and a reproductive tomont. To compare gene expression among Ich life-stages, oligonucleotide microarrays were constructed and utilized. All publicly-available Ich ESTs (~35K) were clustered to generate 9,129 unique consensus sequences represented as probes on custom microarrays produced in coordination with Roche NimbleGen. To facilitate comparative genomic analysis and to potentially increase gene content through cross-hybridization, gene coding sequences of related protozoans Tetrahymena thermophila and Plasmodium falciparum were also added to the microarrays. Gene expression was analyzed in samples taken from each of the three Ich life-stages. The results of this study will add in the understanding of protozoan global gene regulation and biology and should aid in the development of strategies aimed at the control of this important fish parasite. Submitted is a nine chip oligo array design using 385 K Nimblegen arrays. A total of nine microarrays were used for the experiment: three replicates from each of the three Ich life-stages (tomont, trophont, and theront life-stages). Probes were designed using 9,129 unique Ich ESTs (clustered contigs and singletons) as well as 26,273 Tetrahymena thermophila and 5,184 Plasmodium falciparum coding sequences. The probe design strategy was to create 12 60-mer oligonucleotide probes per I. multifiliis sequence, and 10 60-mer oligonucleotide probes for both T. thermophila and P. falciparum sequences. Total RNA was isolated in triplicate from the three life-stages of I. multifiliis and submitted to Nimblegen for labeling, hybridization, and scaning.

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Exploring the potential of a Functional Gene Array (FGA) for fluvial biofilms

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