Project description:Tremella fuciformis is a popular edible fungus with fruiting bodies that can be produced in large quantities at low costs, while it is easy to transform and cultivate as yeast. This makes it an attractive potential bioreactor. Enhanced heterologous gene expression through codon optimization would be useful, but until now codon usage preferences in T. fuciformis remain unknown. The only available genome of a species in the genus Tremella was that of Tremella mesenterica. To precisely determine the preferred codon usage of T. fuciformis we sequenced the genome of strain Tr26 resulting in a 24.5 Mb draft genome with 10,040 predicted genes. 3288 of the derived predicted proteins matched the UniProtKB/Swiss-Prot databases with 40% or more similarity. Corresponding gene models of this subset were subsequently optimized trough repetitive comparison of alternative start codons and selection of best length matching gene models. For experimental confirmation of gene models, 96 random clones from an existing T. fuciformis cDNA library were sequenced, generating 80 complete CDSs. Calculated optimal codons (RSCU and RFCU values) for the 3288 predicted and the 80 cloned CDSs were highly similar, indicating sufficient accuracy of predicted gene models for codon usage analysis. T. fuciformis showed a strong preference for C and then G (C+G; 66.4 %) at the third base pair position of used codons, while average GC content of predicted genes was slightly higher (58.5 %) than he total genome sequence average (55.9%). Most frequently used codons (optimal codons) all ended in C or G except for one (Ter; AGU), and an increased frequency of C ending codons was observed in genes with higher expression levels. Surprisingly, the preferred codon usage in T. fuciformis strongly differed from T. mesenterica (same genus) and C. neoformans (same family). Instead, optimal codon usage was similar to more distant related species such as Ustilago maydis and Neurospora crassa. Despite much higher overall sequence homology between T. fuciformis and T. mesenterica only 7 out of 21 optimal codons were equal, whereas T. fuciformis shared up to 20 out 21 optimal codons with other species. Clearly, codon usage in Tremella can differ largely and should be estimated for individual species. The precise identification of optimal and high expression related codons is therefore an important step in the development of T. fuciformis as a bioreactor system.

Project description:Tremella fuciformis is a popular edible fungus with fruiting bodies that can be produced in large quantities at low costs, while it is easy to transform and cultivate as yeast. This makes it an attractive potential bioreactor. Enhanced heterologous gene expression through codon optimization would be useful, but until now codon usage preferences in T. fuciformis remain unknown. The only available genome of a species in the genus Tremella was that of Tremella mesenterica. To precisely determine the preferred codon usage of T. fuciformis we sequenced the genome of strain Tr26 resulting in a 24.5 Mb draft genome with 10,040 predicted genes. 3288 of the derived predicted proteins matched the UniProtKB/Swiss-Prot databases with 40% or more similarity. Corresponding gene models of this subset were subsequently optimized trough repetitive comparison of alternative start codons and selection of best length matching gene models. For experimental confirmation of gene models, 96 random clones from an existing T. fuciformis cDNA library were sequenced, generating 80 complete CDSs. Calculated optimal codons (RSCU and RFCU values) for the 3288 predicted and the 80 cloned CDSs were highly similar, indicating sufficient accuracy of predicted gene models for codon usage analysis. T. fuciformis showed a strong preference for C and then G (C+G; 66.4 %) at the third base pair position of used codons, while average GC content of predicted genes was slightly higher (58.5 %) than he total genome sequence average (55.9%). Most frequently used codons (optimal codons) all ended in C or G except for one (Ter; AGU), and an increased frequency of C ending codons was observed in genes with higher expression levels. Surprisingly, the preferred codon usage in T. fuciformis strongly differed from T. mesenterica (same genus) and C. neoformans (same family). Instead, optimal codon usage was similar to more distant related species such as Ustilago maydis and Neurospora crassa. Despite much higher overall sequence homology between T. fuciformis and T. mesenterica only 7 out of 21 optimal codons were equal, whereas T. fuciformis shared up to 20 out 21 optimal codons with other species. Clearly, codon usage in Tremella can differ largely and should be estimated for individual species. The precise identification of optimal and high expression related codons is therefore an important step in the development of T. fuciformis as a bioreactor system. Using the Total RNA was extracted from yeast-like cells of T. fuciformis Tr21 in their exponential growth stage, and double-stranded cDNA was synthesized for tag library construction and digital gene expression tag (DGE) sequencing at BGI Tech Solutions Co., Ltd (Shenzhen, China). Image analysis, base calling, extraction of tags, and tag counting were conducted using the Illumina pipeline. Clean tags were mapped to predicted gene models of the draft genome with a mismatch tolerance of 1bp. The number of tags for each CDS was calculated and then normalized to TPM (number of transcripts per million clean tags) digital gene expression (DGE)

Project description:Experiments were done to define the stoichiometry of binding of Cu(I) to metallothionein (MT) and to determine its sites of binding in mixed-metal species. Spectrophotometric titrations of rabbit liver Cd7-MT 2, apoMT, and Cd4-alpha-domain with Cu(I) revealed endpoints of 3-4, 4 and 8, and 4 and 6-7 added Cu(I)/mol of MT for the three species respectively. Observed endpoints depended on conditions of the titration and the wavelength chosen for absorbance measurement. Nevertheless, from metal and sulphydryl analyses of titrated proteins that were pretreated with Chelex-100 to remove metal ions from solution, almost all of the cadmium was displaced from Cd7-MT by the addition of 7 Cu(I)/mol of MT. Similarly, 4 Cu(I)/mol of Cd4-alpha-domain completely displaced bound cadmium. The Cu4-alpha-domain was converted into a Cu6-alpha species upon addition of two equivalents of Cu(I)/mol of alpha-domain. Reaction of Cd7-MT with 7, 12 and 20 Cu/mol of MT, followed by reaction with Chelex resin, generated protein samples in each case with about 7 Cu/mol of MT. 111Cd-NMR analysis of the reaction of 111Cd7-MT with Cu(I) showed that nearly co-operative one-for-one replacement of 111Cd occurred and that the beta-domain cluster reacted before the alpha-domain cluster. Two mixed-metal MTs with Cu to Zn ratios approximating 3 to 4 and 6 to 4 were isolated from calf liver. After substitution of Zn with 111Cd, NMR spectra of each protein showed that 111Cd was confined almost completely to the alpha-domain. By inference, about 3 or 6 Cu were bound in the beta-domain of these proteins. Supporting this segregation of metal ions into domains, reaction of Cu6, Zn4-MT with nitrilotriacetate removed zinc exclusively, whereas reaction of Cu6,Cd4-MT with 4,7-phenylsulphonyl-2,9-dimethyl-1,10-phenanthroline extracted only Cu(I). Proteolytic digestion of both products followed by gel filtration demonstrated that Cu(I) and Cd were bound to fragments of the intact protein. Finally, reaction of rabbit liver 111Cd7-MT 2 with Cu10-MT 2 resulted in interprotein metal exchange in which 111Cd-moved from the beta- to the alpha-domain according to NMR analysis. In contrast with the prevalent view that six Cu(I) bind to each domain of MT, the present results show that Cu(I) binds to MT with a minimum stoichiometry of about 7 Cu(I)/mol of MT and can bind to the alpha-domain with stoichiometries of 4 or 6 Cu(I)/mol of MT. Although MTs interacting with 12 or 20 Cu(I)/mol of MT are less stable than that binding about 7 Cu(I)/mol, it appears that MT can bind Cu(I) in multiple stoichiometries.

Dataset Information

The Fungus Tremella mesenterica Encodes the Longest Metallothionein Currently Known: Gene, Protein and Metal Binding Characterization.

Publications

The Fungus Tremella mesenterica Encodes the Longest Metallothionein Currently Known: Gene, Protein and Metal Binding Characterization.

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