Identification of eukaryotic open reading frames in metagenomic cDNA libraries made from environmental samples - PubMed (original) (raw)

Identification of eukaryotic open reading frames in metagenomic cDNA libraries made from environmental samples

Susan Grant et al. Appl Environ Microbiol. 2006 Jan.

Abstract

Here we describe the application of metagenomic technologies to construct cDNA libraries from RNA isolated from environmental samples. RNAlater (Ambion) was shown to stabilize RNA in environmental samples for periods of at least 3 months at -20 degrees C. Protocols for library construction were established on total RNA extracted from Acanthamoeba polyphaga trophozoites. The methodology was then used on algal mats from geothermal hot springs in Tengchong county, Yunnan Province, People's Republic of China, and activated sludge from a sewage treatment plant in Leicestershire, United Kingdom. The Tenchong libraries were dominated by RNA from prokaryotes, reflecting the mainly prokaryote microbial composition. The majority of these clones resulted from rRNA; only a few appeared to be derived from mRNA. In contrast, many clones from the activated sludge library had significant similarity to eukaryote mRNA-encoded protein sequences. A library was also made using polyadenylated RNA isolated from total RNA from activated sludge; many more clones in this library were related to eukaryotic mRNA sequences and proteins. Open reading frames (ORFs) up to 378 amino acids in size could be identified. Some resembled known proteins over their full length, e.g., 36% match to cystatin, 49% match to ribosomal protein L32, 63% match to ribosomal protein S16, 70% to CPC2 protein. The methodology described here permits the polyadenylated transcriptome to be isolated from environmental samples with no knowledge of the identity of the microorganisms in the sample or the necessity to culture them. It has many uses, including the identification of novel eukaryotic ORFs encoding proteins and enzymes.

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Figures

FIG. 1.

FIG. 1.

Typical results of total RNA extraction from named species and environmental samples electrophoresed on a 1.2% denaturing formaldehyde agarose gel. Lane 1, RNA size markers (Ambion Millennium markers); lane 2, Escherichia coli; lane 3, Saccharomyces cerevisiae; lane 4, Acanthamoeba polyphaga; lanes 5 and 6, activated sludge.

FIG. 2.

FIG. 2.

Effect of storage in RNAlater on the stability of total RNA extracted from Acanthamoeba polyphaga. (A) TBE 1.2% agarose gel. Lane 1, RNA size markers (Millennium markers; Ambion); lanes 2 and 3, total RNA from a fresh culture of Acanthamoeba polyphaga. (B) Same as panel A, but lane 2, total RNA from A. polyphaga stored in RNAlater for 10 days at ambient temperature; lane 3, total RNA from A. polyphaga stored for 10 days at 4°C.

FIG. 3.

FIG. 3.

Results of reverse transcription and LD-PCR on RNA from different sources to produce cDNAs run out on TBE 1.2% agarose gels. (A) Lane 1, 1-kb DNA ladder (Invitrogen); lane 2, negative control; lane 3, product from the human placental mRNA provided as a control in the Smart cDNA Library construction kit. (B) Lane 1, 1-kb ladder; lanes 2 to 5, product from total RNA extracted from Acanthamoeba polyphaga. (C) Same as panel B, showing product from the LP4 Chinese algal mat. (D) Same as panel B, showing product from activated sludge total RNA.

FIG. 4.

FIG. 4.

18S rRNA gene amplification from algal mat samples and activated sludge. (A) Lane 1, 1-kb ladder; lane 2, negative control; lane 3, Acanthamoeba polyphaga positive control; lane 4, product from activated sludge. (B) Lane 1, 1-kb ladder; lane 2, negative control; lane 3, Acanthamoeba polyphaga positive control; lane 4, algal mat LP4; lanes 5 and 6, product from two different DNA extracts from algal mat TC2.

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