Translational control of recombinant human acetylcholinesterase accumulation in plants

<p>Abstract</p> <p>Background</p> <p>Codon usage differences are known to regulate the levels of gene expression in a species-specific manner, with the primary factors often cited to be mRNA processing and accumulation. We have challenged this conclusion by expressing the human acetylcholinesterase coding sequence in transgenic plants in its native GC-rich sequence and compared to a matched sequence with (dicotyledonous) plant-optimized codon usage and a lower GC content.</p> <p>Results</p> <p>We demonstrate a 5 to 10 fold increase in accumulation levels of the "synaptic" splice variant of human acetylcholinesterase in <it>Nicotiana benthamiana </it>plants expressing the optimized gene as compared to the native human sequence. Both transient expression assays and stable transformants demonstrated conspicuously increased accumulation levels. Importantly, we find that the increase is not a result of increased levels of acetylcholinesterase mRNA, but rather its facilitated translation, possibly due to the reduced energy required to unfold the sequence-optimized mRNA.</p> <p>Conclusion</p> <p>Our findings demonstrate that codon usage differences may regulate gene expression at different levels and anticipate translational control of acetylcholinesterase gene expression in its native mammalian host as well.</p

Osiris: accessible and reproducible phylogenetic and phylogenomic analyses within the Galaxy workflow management system

BACKGROUND: Phylogenetic tools and ‘tree-thinking’ approaches increasingly permeate all biological research. At the same time, phylogenetic data sets are expanding at breakneck pace, facilitated by increasingly economical sequencing technologies. Therefore, there is an urgent need for accessible, modular, and sharable tools for phylogenetic analysis. RESULTS: We developed a suite of wrappers for new and existing phylogenetics tools for the Galaxy workflow management system that we call Osiris. Osiris and Galaxy provide a sharable, standardized, modular user interface, and the ability to easily create complex workflows using a graphical interface. Osiris enables all aspects of phylogenetic analysis within Galaxy, including de novo assembly of high throughput sequencing reads, ortholog identification, multiple sequence alignment, concatenation, phylogenetic tree estimation, and post-tree comparative analysis. The open source files are available on in the Bitbucket public repository and many of the tools are demonstrated on a public web server (http://galaxy-dev.cnsi.ucsb.edu/osiris/). CONCLUSIONS: Osiris can serve as a foundation for other phylogenomic and phylogenetic tool development within the Galaxy platform

Translational control of recombinant human acetylcholinesterase accumulation in plants-3

<p><b>Copyright information:</b></p><p>Taken from "Translational control of recombinant human acetylcholinesterase accumulation in plants"</p><p>http://www.biomedcentral.com/1472-6750/7/27</p><p>BMC Biotechnology 2007;7():27-27.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1913049.</p><p></p>. cDNA prepared from three lines of plants (1) and two lines of plants (2) were subjected to PCR amplification using the primers oTM089 and oTM049 and then resolved by agarose gel electrophoresis. Primers were selected to amplify the region potentially subjected to alternative splicing. Unspliced transcripts are expected to yield 1.1 kb fragment. If either of the two potential 5'- and 3'-intron splice sites were extensively utilized in plants harboring , shorter fragments of ~0.9 kb and ~0.5 kb are expected to be preferentially amplified. However, the full-length species clearly dominates. () Correlation between transcript accumulation and AChE enzyme activity. Transcripts levels of optimized (pTM092) and non-optimized (pTM050) measured by quantitative real time RT-PCR and were scored relative to 18S rRNA. Probes were designed for the two common regions of the two types of transcripts – () the 5'-UTR (TEV leader) and () the 3'-UTR (VSP terminator, upstream of the poly-A tail). Data obtained for multiple replicates was averaged and correlated with the respective AChE activity (normalized to the maximum level). When probed by the 5'-UTR probe, the correlation was highly significant with r= 0.72 (< 0.0001) for the non-optimized sequence and r= 0.61 (= 0.0005) for the optimized sequence. When probed by the 3'-UTR probe, the correlation was significant for the non-optimized sequence with r= 0.48 (= 0.0122) and highly significant with r= 0.49 (< 0.0001) for the optimized sequence. Inserts show scatter plots demonstrating the distribution of transcript levels in plants expressing and constructs. Each plant is depicted by a symbol and the mean ± S.D. are plotted. The groups are not statistically different by an unpaired t test (5'-UTR probe: > ; 3'-UTR probe: > )

Translational control of recombinant human acetylcholinesterase accumulation in plants-5

<p><b>Copyright information:</b></p><p>Taken from "Translational control of recombinant human acetylcholinesterase accumulation in plants"</p><p>http://www.biomedcentral.com/1472-6750/7/27</p><p>BMC Biotechnology 2007;7():27-27.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1913049.</p><p></p> . Averages of AChE activity of 6 replicates were normalized to that of to obtain the fold increase values (+ SEM) shown

Translational control of recombinant human acetylcholinesterase accumulation in plants-7

<p><b>Copyright information:</b></p><p>Taken from "Translational control of recombinant human acetylcholinesterase accumulation in plants"</p><p>http://www.biomedcentral.com/1472-6750/7/27</p><p>BMC Biotechnology 2007;7():27-27.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1913049.</p><p></p>codon adaptiveness () was scored for (pink), (blue), and the highly-expressed plant gene (red). The CAI values of each (averaged over the entire gene) are plotted as broken lines. Note that optimization increased the CAI value of the gene, bringing it close to the CAI value of the gene, which is even for this highly expressed gene lower than the theoretical maximal value of 1. The GC content is scored for 100-base segments centering at each of the indicated bases of the and mRNA (taking into account the common TEV leader). Broken lines – the GC content of the entire coding regions

Translational control of recombinant human acetylcholinesterase accumulation in plants-2

<p><b>Copyright information:</b></p><p>Taken from "Translational control of recombinant human acetylcholinesterase accumulation in plants"</p><p>http://www.biomedcentral.com/1472-6750/7/27</p><p>BMC Biotechnology 2007;7():27-27.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1913049.</p><p></p>M092 driving the expression of . pTM256 driving mosaic . pTM257 driving the expression of mosaic . 35S, CaMV 35S promoter; T, TEV leader; V, VSP terminator. Arrow heads, positions of oligonucleotides used in this for making probes, genomic PCR and RT-PCR (numbers refer to Table 2). (Transient expression assays in -infiltrated leaves of plants. 1, ; 2, . Averages of 6 replicates (means ± SEM) are shown. () plants were stably transformed to express (1) or (2) . Individual lines were screened at least 2 times. Averages of all lines (presented in Panel D) with detectible levels of AChE (means ± SEM) are shown. () AChE accumulation levels in individual genomic-PCR positive plant lines expressing . Top inserts: AChE accumulation levels in individual plant lines expressing variants of : pTM167, pTM165 and pTM164. Bottom inserts: AChE accumulation levels in individual plant lines expressing variants of : pTM195, pTM197 and pTM156. Broken lines illustrate the maximal level of expression obtained with any of the lines. DNA fragments were PCR amplified from total DNA isolated from the indicated plant lines with specific primers (oTM126 and oTM047) and were resolved by agarose gel electrophoresis (denoted PCR). () Cumulative frequency distribution of the AChE expression data of lines shown in Panel D (high to low) can be fitted with the following logarithmic regression equations: , y = -22.967Ln (x) + 107.63 (r= 0.90); : y = -15.013Ln (x) + 102.99 (r= 0.98)

Translational control of recombinant human acetylcholinesterase accumulation in plants-4

<p><b>Copyright information:</b></p><p>Taken from "Translational control of recombinant human acetylcholinesterase accumulation in plants"</p><p>http://www.biomedcentral.com/1472-6750/7/27</p><p>BMC Biotechnology 2007;7():27-27.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1913049.</p><p></p>e presence of the cytosolic protein synthesis inhibitor cycloheximide (A, +CHX) or in its absence (B). Plotted are means ± SEM of individual explant samples (= 1–8). Non-linear regression analysis was performed using the Prism software with the resultant equations. oAChES + CHX: y = 103e(R= 0.78); oAChES: y = 103e(r= 0.36); hAChES + CHX: y = 98e(r= 0.6123); hAChES: y = 95e(r= 0.4463). The differences between the regression lines obtained in the presence of CHX are not significant (unpaired test, = 0.195) while all other combinations are highly significant (< 0.0001)