Differential use of termination codons in ciliated protozoa.

Harper, D. Scott; Jahn, Carolyn L.

doi:10.1073/pnas.86.9.3252

Cited by 69 publications

(27 citation statements)

References 32 publications

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“…Similarly, differences in the usage of termination codons have been observed in ciliated protozoa (13), and in some ciliates, stop codons are reassigned to sense codons (19). Clearly, the appropriate code can be selected when sequencing clones from a known organism, but choice is more problematic for environmental isolates.…”

Section: Resultsmentioning

confidence: 99%

Identification of Eukaryotic Open Reading Frames in Metagenomic cDNA Libraries Made from Environmental Samples

Grant

Cowan

et al. 2006

Appl Environ Microbiol

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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°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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Section: Resultsmentioning

confidence: 99%

Identification of Eukaryotic Open Reading Frames in Metagenomic cDNA Libraries Made from Environmental Samples

Grant

Cowan

et al. 2006

Appl Environ Microbiol

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“…This assumption was based on sequence analyses of genes in four genera: Paramecium (1, 2), Tetrahymena (3-7), Stylonychia (8), and Oxytricha (9). Recently this view was challenged by the finding that in Euplotes crassus (10) and Euplotes raikovi (11), UAA is used as a termination codon, indicating that the Euplotids differ in this respect from other ciliates. Here we report on the cDNA and amino acid sequence of pheromone 3 of Euplotes octocarinatus.…”

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UGA is translated as cysteine in pheromone 3 of Euplotes octocarinatus.

Meyer

Schmidt

Plümper

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

159

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Pheromone 3 mRNA of the ciliate Euplotes octocarihatus contains three in-frame UGA codons that are translated as cysteines. This was revealed from cDNA sequencing and from plasma desorption mass spectrometry of cleaved pheromone 3 in connection with pyridylethylation of the fragments. N-terminal sequence analysis of carboxymethylated protein confirmed this conclusion for the first of the three UGA codons. Besides UGA the common cysteine codons UGU and UGC are also used to encode cysteine. UAA functions as a termination codon. Preparation of RNA. Total RNA was prepared by disruption of 1-3 x 107 cells in 8 M urea/4 M LiCl in a PotterElvehjem homogenizer, followed by precipitation on ice overnight. RNA was collected by centrifugation, dissolved in 10 mM Mops, pH 7.5/0.5% SDS, and extracted three times with phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform/isoamyl alcohol (24:1). Total RNA was then precipitated by addition of 0.1 volume of 4 M LiCl and 2.5 volumes of absolute ethanol.Poly(A)+ RNA was prepared by affinity chromatography on oligo(dT)-cellulose (Bethesda Research Laboratories) as recommended by the supplier with the exception that Mops was used as the buffer instead of Tris. Poly(A)+ RNA was precipitated by the addition of LiCI and ethanol as described above and redissolved in water. Quantity and quality were determined spectrophotometrically by measuring absorption at 260 and 280 nm (14).cDNA Synthesis and Cloning. The cDNA library was constructed (15) in the vector AgtlO. The cDNA was treated with S1 nuclease and ligated with EcoRI linkers prior to its insertion into the EcoRI site of the vector. The pheromone 3 gene was identified by plaque hybridization with the synthetic oligodeoxynucleotide 5'-GTRTANGGYTCYTCCCA-3', corresponding to the N terminus of the secreted pheromone, and was isolated by standard techniques (14).DNA Sequencing. Eight positively hybridizing plaques were obtained from 105 transformants. Five of them were further subcloned for sequencing by the dideoxy chain-termination method. Their nucleotide sequences were determined from double-stranded and single-stranded templates (pUC12, pT7T3, M13mpl8, and M13mp19 as sequencing vectors) according to the sequencing strategy outlined in Fig. 1 1To whom reprint requests should be addressed.

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“…Alternatively, it has been suggested that a second unusual genetic feature of Euplotes, stop-codon reassignment, is involved in slowing translation and promoting the frameshifts (22). Euplotids have reassigned the UGA stop codon of the universal code so that it now encodes cysteine (16,28). This has occurred, in part, as a result of changes to the single eukaryotic translation release factor 1 protein (eRF1) so that it no longer recognizes the UGA stop codon (9, 21, 34a).…”

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Sequencing of Random Euplotes crassus Macronuclear Genes Supports a High Frequency of +1 Translational Frameshifting

Klobutcher

2005

Eukaryot Cell

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Programmed translational frameshifts have been identified in genes from a broad range of organisms, but typically only a very few genes in a given organism require a frameshift for expression. In contrast, a recent analysis of gene sequences available in GenBank from ciliates in the genus Euplotes indicated that >5% required one or more ؉1 translational frameshifts to produce their predicted protein products. However, this sample of genes was nonrandom, biased, and derived from multiple Euplotes species. To test whether there truly is an abundance of frameshift genes in Euplotes, and to more accurately assess their frequency, we sequenced a random sample of 25 cloned genes/macronuclear DNA molecules from Euplotes crassus. Three new candidate ؉1 frameshift genes were identified in the sample that encode a membrane occupation and recognition nexus (MORN) repeat protein, a C 2 H 2 -type zinc finger protein, and a Ser/Thr protein kinase. Reverse transcription-PCR analyses indicate that all three genes are expressed in vegetatively proliferating cells and that the mRNAs retain the requirement of a frameshift. Although the sample of sequenced genes is relatively small, the results indicate that the frequency of genes requiring frameshifts in E. crassus is between 3.7% and 31.7% (at a 95% confidence interval). The current and past data also indicate that frameshift sites are found predominantly in genes that likely encode nonabundant proteins in the cell.During the translation of an mRNA, a shift in reading frame is usually a catastrophic event, often resulting in a truncated and/or nonfunctional protein. Translational frameshifting is infrequent during the translation of most mRNAs (Ͻ5 ϫ 10 Ϫ5 frameshifts per codon translated [25]), but a number of mRNAs require a frameshift to express a functional protein and appear to have evolved to stimulate frameshifting. Such programmed translational frameshifting (reviewed in references 13 and 32) is relatively rare but is phylogenetically widespread, as examples are known in prokaryotes, eukaryotes, and a number of mobile genetic elements. Sequence elements within the mRNA facilitate programmed frameshifting, and these typically reside at the site of the frameshift, but more distally located sequences can also be involved.A number of genes in ciliated protozoa of the genus Euplotes (class Spirotrichea) have been identified that appear to require a ϩ1 translational frameshift to produce their protein products. The putative ϩ1-frameshift genes encode the regulatory subunit of cyclic AMP-dependent protein kinase and a nuclear protein kinase of Euplotes octocarinatus (39, 40), a La motif protein (p43) in Euplotes aediculatus (1), the Euplotes crassus Tec2 transposon ORF2 protein (11,18), and the reverse transcriptase subunits of telomerase (TERT) in three euplotid species (20,29,42). Since the complete sequences of less than 100 Euplotes genes have been determined, it appears that frameshifting is unusually common in euplotids, with perhaps Ͼ5% of genes requiring a frameshift for ...

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Differential use of termination codons in ciliated protozoa.

Cited by 69 publications

References 32 publications

Identification of Eukaryotic Open Reading Frames in Metagenomic cDNA Libraries Made from Environmental Samples

Identification of Eukaryotic Open Reading Frames in Metagenomic cDNA Libraries Made from Environmental Samples

UGA is translated as cysteine in pheromone 3 of Euplotes octocarinatus.

Sequencing of Random Euplotes crassus Macronuclear Genes Supports a High Frequency of +1 Translational Frameshifting

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