Gene discovery and expression profile analysis through sequencing of expressed sequence tags from different developmental stages of the chytridiomycete Blastocladiella emersonii

Abstract

Blastocladiella emersonii is an aquatic fungus of the chytridiomycete class which diverged early from the fungal lineage and is notable for the morphogenetic processes which occur during its life cycle. Its particular taxonomic position makes this fungus an interesting system to be considered when investigating phylogenetic relationships and studying the biology of lower fungi. To contribute to the understanding of the complexity of the B. emersonii genome, we present here a survey of expressed sequence tags (ESTs) from various stages of the fungal development. Nearly 20,000 cDNA clones from 10 different libraries were partially sequenced from their 5' end, yielding 16,984 high-quality ESTs. These ESTs were assembled into 4,873 putative transcripts, of which 48% presented no matches with existing sequences in public databases. As a result of Gene Ontology (GO) project annotation, 1,680 ESTs (35%) were classified into biological processes of the GO structure, with transcription and RNA processing, protein biosynthesis, and transport as prevalent processes. We also report full-length sequences, useful for construction of molecular phylogenies, and several ESTs that showed high similarity with known proteins, some of which were not previously described in fungi. Furthermore, we analyzed the expression profile (digital Northern analysis) of each transcript throughout the life cycle of the fungus using Bayesian statistics. The in silico approach was validated by Northern blot analysis with good agreement between the two methodologies.

FIG. 1.

Summary of BLASTX analysis of B. emersonii predicted proteins. The proportions of B. emersonii predicted proteins with BLASTX best hits (E values of ≤10⁻⁶) to fungal and nonfungal eukaryotes and bacteria are indicated. The fungal and nonfungal eukaryotes with the highest percentages of BLASTX best hits to B. emersonii predicted proteins are shown. Abbreviations: M. grisea, Magnaporthe grisea; A. nidulans, Aspergillus nidulans; R. norvegicus, Rattus norvegicus; X. laevis, Xenopus laevis; D. melanogaster, Drosophila melanogaster; A. gambiae, Anopheles gambiae; A. thaliana, Arabidopsis thaliana; M. musculus, Mus musculus; H. sapiens, Homo sapiens.

FIG. 2.

Fragments of alignments between EF-1α (A) and enolase (B) sequences from B. emersonii and those of selected organisms. (A) A 12-aa insertion shared by animals and fungi (B. emersonii has one extra amino acid) and three insertions (8, 5, and 4 aa) shared only by B. emersonii and M. brevicollis. (B) Four gaps (1-aa deletion, 2-aa insertion, 1-aa deletion, and 5-aa deletion) shared by animals and fungi. B. emersonii has two extra amino acids in the 2-aa insertion. Alignments were made with the Clustal W program and manually revised. The numbers above the alignments indicate B. emersonii sequence positions. Abbreviations: *, residues identical in all sequences; :, conserved substitutions; ., semiconserved substitutions; Ch, choanoflagellates; Fu, fungi; Me, metazoa; Vp, viridiplantae; Eu, euglenozoa; Mb, M. brevicollis (gi 16554295); Be, B. emersonii (gi number pending); Um, U. maydis (gi 49068500); Sc, S. cerevisiae (gi 171454); Hs, Homo sapiens (gi 15421129, gi 338694); Dm, Drosophila melanogaster (gi 7915, gi 158738); Le, Lycopersicon esculentum (gi 295810, gi 19280); Zm, Zea mays (gi 2282584, gi:22272); Eg, Euglena gracilis (gi 119148, gi 18438).

FIG. 3.

Abundance profiles of 109 different transcripts during the B. emersonii life cycle. Distinct profiles were selected from digital Northern analysis data and subjected to hierarchical clustering. The abundance (from 0 to 0.01) was estimated by considering the number of ESTs sequenced for a given transcript divided by the total number of ESTs sequenced in each library as follows: 1, E30; 2, E60; 3, E90; 4, E120; 5, ZSP; 6, G30; 7, G60; 8, G90; 9, G120. The library names are as given in Materials and Methods. The first column shows the identification number of selected contigs from digital Northern analysis. The descriptions in parentheses are the best hits for each contig using BLASTX against the Swiss-Prot database. Transcripts highlighted in orange have the GO classifications GO:0006508 and GO:0006457, which correspond to the biological processes proteolysis or peptidolysis and protein folding, respectively; in blue are transcripts classified as GO:0006412, which corresponds to protein biosynthesis; in red are transcripts classified as GO:0006334, which corresponds to chromosome structure. *, genes manually annotated to GO:0006508 and GO:0006457.

FIG. 4.

Expression patterns of selected transcripts differentially expressed during the B. emersonii life cycle. The top portions of each panel shows autoradiograms of Northern blot experiments using total RNA isolated from synchronized cells at the indicated stages of the life cycle. The bottom portions show line plots comparing data obtained from the Northern blot (□) with data obtained from the digital Northern analysis (▪). The number in the left top corner of each plot corresponds to the Pearson correlation. Putative proteins encoded by each transcript are as follows: 40S ribosomal protein S9 (A), histone H2A (B), unknown (C), elongation factor 1α long form (D), unknown (E), unknown (F), calmodulin (G), and unknown (H). The results obtained by densitometric analysis of Northern blot hybridization signals confirm the in silico expression profiles.