Expression of Nucleus‐Encoded Genes for Chloroplast Proteins in the Flagellate <i>Euglena gracilis</i>

Vesteg, Matej; Vacula, Rostislav; Burey, Suzanne C.; Löffelhardt, Wolfgang; Drahovská, Hana; Martin, William; Krajčovič, Juraj

doi:10.1111/j.1550-7408.2008.00383.x

Cited by 22 publications

(26 citation statements)

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“…Genomic DNA was isolated as in the protocol described earlier using phenolchloroform method [29]. RNA extraction and mRNA isolation were carried out as described previously [29,30]. cDNA synthesis was performed with oligo(dT) and random hexanucleotide primers using ImProm-II Reverse Transcription System (Promega, Madison, WI, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Hereafter, total RNA means the full complement of mRNAs for individual genes and polyadenylated RNA means the polyadenylated fraction thereof. We used the expression of nuclear gene PsbO to normalize the calculations, because our previous studies have revealed that its mRNA level is the same in the light-and dark-grown E. gracilis [30,32].…”

Section: Quantitative Pcrmentioning

confidence: 99%

“…PCR was performed as described earlier [29,30] except the annealing temperature of 60°C instead of 58°C. In the first set of PCR reactions, the 26 bp-long SL sequence present at the 5 0 -end of E. gracilis nuclear mRNAs was used as a forward primer (SL).…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

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A small portion of plastid transcripts is polyadenylated in the flagellate Euglena gracilis

et al. 2014

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Edited by Ulf-Ingo FlüggeKeywords: Plastid EST Euglenozoa Polyadenylation Quantitative PCR Trans-splicing a b s t r a c t Euglena gracilis possesses secondary plastids of green algal origin. In this study, E. gracilis expressed sequence tags (ESTs) derived from polyA-selected mRNA were searched and several ESTs corresponding to plastid genes were found. PCR experiments failed to detect SL sequence at the 5 0 -end of any of these transcripts, suggesting plastid origin of these polyadenylated molecules. Quantitative PCR experiments confirmed that polyadenylation of transcripts occurs in the Euglena plastids. Such transcripts have been previously observed in primary plastids of plants and algae as low-abundance intermediates of transcript degradation. Our results suggest that a similar mechanism exists in secondary plastids. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. IntroductionEuglena gracilis is a fresh-water photosynthetic flagellate belonging to the order Euglenida and to the protist phylum Euglenozoa [1,2]. This phylum includes also the orders Kinetoplastida, Diplonemida and Symbiontida [3,4], which comprise exclusively heterotrophic species. Most euglenid species are free-living heterotrophic flagellates, but some of them possess secondary plastids that arose via secondary endosymbiosis of a green alga [5]. Phylogenetic analysis of plastid genome sequences revealed that euglenid plastids are derived from a Pyramimonas-related prasinophyte alga [6].A common euglenozoan feature is the processing of primary nuclear transcripts by spliced leader (SL) RNA-mediated trans-splicing [7]. This process includes replacement of the 5 0 -end of premRNA by the 5 0 -end of SL-RNA, donating identical 5 0 -termini to the mRNA molecules. Similar to nuclear cis-splicing, trans-splicing process is also mediated by spliceosomes, but a Y-branch intron structure is formed instead of a lariat [8]. The only currently known nuclear mRNA lacking the SL sequence in E. gracilis is that of the fibrillarin gene [9]. Since SL-trans-splicing does not occur in organelles (mitochondria, plastids), the presence (or absence) of an SL sequence at the 5 0 -end of a euglenozoan mRNA is diagnostic for its synthesis in the nucleus (or organelles, respectively).E. gracilis cell possesses approximately eight secondary plastids bounded by three membranes [10,11]. The plastid genome of this species is circular and comprises 143.17 kb. It contains 96 protein and RNA gene loci [12], group II and III introns, and twintrons (i.e. introns within introns) [13].The evolutionary transition from an endosymbiont to the plastid organelle was accompanied by a loss of many genes and gene transfer from the endosymbiont genome(s) to the host genome [14]. Gene transfer from plastids and mitochondria is an ongoing process, as it has been demonstrated in animals, plants, fungi as well as protists [15,16].Nuclear copies of plastid DNA (NUPT) can be found in a variety of species [17]. However, some species, e.g. the ...

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Section: Methodsmentioning

confidence: 99%

Section: Quantitative Pcrmentioning

confidence: 99%

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A small portion of plastid transcripts is polyadenylated in the flagellate Euglena gracilis

et al. 2014

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“…9.1; Kishore and Schwartzbach 1992b;Weiss et al 1988Weiss et al , 1992. pLHCPII synthesis was undetectable in the bleached mutants, W 3 BUL and W 10 BSmL, which have lost most if not all of their plastid genome even though the level of pLHCPII mRNA was similar to the level in dark grown wild type Euglena (Kishore and Schwartzbach 1992a, b;Schiff et al 1991b;Vesteg et al 2009). A major fraction of pLHCPII mRNA was associated with polysomes in the bleached mutants, W 3 BUL and W 10 BSmL (Kishore and Schwartzbach 1992b).…”

Section: Does Light Regulate Nuclear Gene Expression At the Transcripmentioning

confidence: 93%

Photo and Nutritional Regulation of Euglena Organelle Development

Schwartzbach

2017

Advances in Experimental Medicine and Biology

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Euglena can use light and CO, photosynthesis, as well as a large variety of organic molecules as the sole source of carbon and energy for growth. Light induces the enzymes, in this case an entire organelle, the chloroplast, that is required to use CO as the sole source of carbon and energy for growth. Ethanol, but not malate, inhibits the photoinduction of chloroplast enzymes and induces the synthesis of the glyoxylate cycle enzymes that comprise the unique metabolic pathway leading to two carbon, ethanol and acetate, assimilation. In resting, carbon starved cells, light mobilizes the degradation of the storage carbohydrate paramylum and transiently induces the mitochondrial proteins required for the aerobic metabolism of paramylum to provide the carbon and energy required for chloroplast development. Other mitochondrial proteins are degraded upon light exposure providing the amino acids required for the synthesis of light induced proteins. Changes in protein levels are due to increased and decreased rates of synthesis rather than changes in degradation rates. Changes in protein synthesis rates occur in the absence of a concomitant increase in the levels of mRNAs encoding these proteins indicative of photo and metabolic control at the translational rather than the transcriptional level. The fraction of mRNA encoding a light induced protein such as the light harvesting chlorophyll a/b binding protein of photosystem II, (LHCPII) associated with polysomes in the dark is similar to the fraction associated with polysomes in the light indicative of photoregulation at the level of translational elongation. Ethanol, a carbon source whose assimilation requires carbon source specific enzymes, the glyoxylate cycle enzymes, represses the synthesis of chloroplast enzymes uniquely required to use light and CO as the sole source of carbon and energy for growth. The catabolite sensitivity of chloroplast development provides a mechanism to prioritize carbon source utilization. Euglena uses all of its resources to develop the metabolic capacity to utilize carbon sources such as ethanol which are rarely in the environment and delays until the rare carbon source is no longer available forming the chloroplast which is required to utilize the ubiquitous carbon source, light and CO.

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“…This process is obligatory for these organisms, because mRNAs obtain cap from 5'-end of SL-RNA via this process and the cap is necessary for translation. This process does not allow effective regulation of gene expression at the level of transcription initiation, because SL-RNAs and pre-mRNAs are transcribed from different genomic regions (Martínez-Calvillo et al 2010;Vesteg et al 2009). Therefore, the process is energetically costly in comparison to the effective regulatory condition seen in prokaryotes and many model eukaryotes.…”

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Selective forces for the origin of spliceosomes

2012

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It has been proposed that eukaryotic spliceosomes evolved from bacterial group II introns via constructive neutral changes. However, a more likely interpretation is that spliceosomes and group II introns share a common undefined RNA ancestor--a proto-spliceosome. Although, the constructive neutral evolution may have probably played some roles in the development of complexity including the evolution of modern spliceosomes, in fact, the origin, losses and the retention of spliceosomes can be explained straight-forwardly mainly by positive and negative selection: (1) proto-spliceosomes evolved in the RNA world as a mechanism to excise functional RNAs from an RNA genome and to join non-coding information (ancestral to exons) possibly designed to be degraded. (2) The complexity of proto-spliceosomes increased with the invention of protein synthesis in the RNP world and they were adopted for (a) the addition of translation signal to RNAs via trans-splicing, and for (b) the exon-shuffling such as to join together exons coding separate protein domains, to translate them as a single unit and thus to facilitate the molecular interaction of protein domains needed to be assembled to functional catalytic complexes. (3) Finally, the spliceosomes were adopted for cis-splicing of (mainly) non-coding information (contemporary introns) to yield translatable mRNAs. (4) Spliceosome-negative organisms (i.e., prokaryotes) have been selected in the DNA-protein world to save a lot of energy. (5) Spliceosome-positive organisms (i.e., eukaryotes) have been selected, because they have been completely spliceosome-dependent.

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Expression of Nucleus‐Encoded Genes for Chloroplast Proteins in the Flagellate Euglena gracilis

Cited by 22 publications

References 57 publications

A small portion of plastid transcripts is polyadenylated in the flagellate Euglena gracilis

A small portion of plastid transcripts is polyadenylated in the flagellate Euglena gracilis

Photo and Nutritional Regulation of Euglena Organelle Development

Selective forces for the origin of spliceosomes

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