BackgroundTrichoderma reesei is an organism involved in degradation of (hemi)cellulosic biomass. Consequently, the corresponding enzymes are commonly used in different types of industries, and recently gained considerable importance for production of second-generation biofuel. Many industrial T. reesei strains currently in use are derived from strain Rut-C30, in which cellulase and hemicellulase expression is released from carbon catabolite repression. Nevertheless, inducing substances are still necessary for a satisfactory amount of protein formation.ResultsHere, we report on a T. reesei strain, which exhibits a very high level of xylanase expression regardless if inducing substances (e.g. D-xylose, xylobiose) are used. We found that a single point mutation in the gene encoding the Xylanase regulator 1 (Xyr1) is responsible for this strong deregulation of endo-xylanase expression and, moreover, a highly elevated basal level of cellulase expression. This point mutation is localized in a domain that is common in binuclear zinc cluster transcription factors. Only the use of sophorose as inducer still leads to a slight induction of cellulase expression. Under all tested conditions, the formation of cbh1 and cbh2 transcript level strictly follows the transcript levels of xyr1. The correlation of xyr1 transcript levels and cbh1/cbh2 transcript levels and also their inducibility via sophorose is not restricted to this strain, but occurs in all ancestor strains up to the wild-type QM6a.ConclusionsEngineering a key transcription factor of a target regulon seems to be a promising strategy in order to increase enzymes yields independent of the used substrate or inducer. The regulatory domain where the described mutation is located is certainly an interesting research target for all organisms that also depend so far on certain inducing conditions.
Mitochondrial ribosomes contain bacterial-type proteins reflecting their endosymbiotic heritage, and a subset of these genes is retained within the mitochondrion in land plants. Variation in gene location is observed, however, because migration to the nucleus is still an ongoing evolutionary process in plants. To gain insights into adaptation events related to successful gene transfer, we have compiled data for bacterial-origin mitochondrial-type ribosomal protein genes from the completely sequenced Arabidopsis and rice genomes. Approximately 75% of such nuclear-located genes encode amino-terminal extensions relative to their Escherichia coli counterparts, and of that set, only about 30% have introns at (or near) the junction in support of an exon shuffling-type recruitment of upstream expression/targeting signals. We find that genes that were transferred to the nucleus early in eukaryotic evolution have, on average, about twofold higher density of introns within the core ribosomal protein sequences than do those that moved to the nucleus more recently. About 20% of such introns are at positions identical to those in human orthologs, consistent with their ancestral presence. Plant mitochondrial-type ribosomal protein genes have dispersed chromosomal locations in the nucleus, and about 20% of them are present in multiple unlinked copies. This study provides new insights into the evolutionary history of endosymbiotic bacterial-type genes that have been transferred from the mitochondrion to the nucleus.
We have compared the RNA editing status of wheat mitochondrial unspliced precursor transcripts with spliced RNAs, focusing in particular on exon editing sites located very close to intron/exon junctions. Using direct sequencing of reverse transcriptase-PCR products to assess C-to-U editing in various RNA populations for the respiratory chain genes nad2, nad4, nad5, nad7, and cox2, we observed that candidate exon sites immediately upstream or downstream of introns remained unedited in the presence of the adjacent intron, whereas sites further away were typically partially (or completely) edited in precursor molecules. The 'late' editing of exon sites adjacent to splice junctions is consistent with access of the editing machinery being sterically hindered by the intron or alternatively by an editing recognition element being created by the exon/exon structure. When we examined RNA isolated from the embryo-toseedling stages of wheat development, we found that fully spliced messenger RNAs showed editing at the expected sites in all stages, including dormant seeds. In contrast, we observed that cox2 precursors were less completely edited in RNA populations from 0 to 24 h postimbibition embryos than in 6-day-old seedlings not only at splice junctions but also at other exon sites, consistent with more efficient coupling between editing and transcription during later stages of development.
Mitochondrial genes for ribosomal proteins undergo relatively frequent transfer to the nucleus during plant evolution, and when migration is successful the mitochondrial copy becomes redundant and can be lost. We have examined the status of the mitochondrial rps19 gene for ribosomal protein S19 in closely related cereals. In oat, the mitochondrial rps19 reading frame is blocked by a premature termination codon and lacks abundant transcripts, whereas in the mitochondria of wheat and rye rps19 is a 5'-truncated pseudogene which is co-transcribed with the downstream nad4L gene. In barley and maize, rps19 sequences are completely absent from the mitochondrion. All five of these cereals differ from rice, in which an intact, transcriptionally active mitochondrial rps19 gene is found, and this is preceded by rpl2 in an organization reminiscent of that seen in bacteria. Based on EST sequence data for maize, barley and wheat, it can be inferred that a functional rps19 gene was transferred to the nucleus prior to the divergence of the maize and rice lineages (approximately 50 million years ago), and the present-day nuclear copies encode an N-terminal sequence related to the mitochondrial targeting signal of Hsp70 (heat shock protein) in cereals. Subsequent evolutionary events have included independent losses of the mitochondrial copies in the barley and maize lineages. In the rice lineage, on the other hand, the nuclear copy was lost. This is reflected in the persistence of the mitochondrial rps19 after a period during which rps19 genes coexisted in both compartments. These observations illustrate the dynamic nature of the location and structure of genes for mitochondrial ribosomal proteins in flowering plants.
We have examined precursor and processed transcripts arising from the wheat mitochondrial ccmFN-rps1 region, which encodes a cytochrome c biogenesis component and S1 ribosomal protein, for the embryo-to-seedling stages of development. Northern analysis revealed 3.2-kb ccmFN-rps1 precursors, 2.6-kb bicistronic mRNA and 0.7-kb monocistronic rps1 transcripts, although their relative abundances were seen to shift during development. The 3.2-kb transcript levels peak during the 12-h to 2-day period, whereas 2.6-kb transcripts continue to increase during seedling growth, consistent with the newly-synthesized RNAs being more efficiently processed in later developmental stages. The 3.2-kb ccmFN-rps1 precursors consist of primary transcripts and 5'-processed RNAs based on pyrophosphatase-treated circular-RT-PCR analysis, whereas the 5' termini of 2.6-kb transcripts appear to be generated by endonucleolytic cleavage. The 0.7-kb rps1 transcripts are abundant during early germination but not in the seedlings; their 5' ends are heterogeneous and most of them lack the expected initiation codon. Notably all three size classes of RNAs share similar 3' termini. The 2.6-kb ccmFN-rps1 mRNAs exhibited full C-to-U editing at the sites examined, whereas the other two categories were slightly under-edited. A subset of all three-sized transcripts possessed short stretches of non-encoded adenosines, thus adding another layer of complexity to RNA level events in plant mitochondria.
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