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" Structure and biosynthesis of yeast cell wall mannan "
To better understand the entire process of galactomannan biosynthesis and to begin to identify components of the transcriptional regulatory network controlling this process, deep EST profiling of fenugreek seed endosperm was performed (). In this system, large quantities of galactomannans accumulate over a relatively short time frame, formed from sucrose that enters the developing seed (). Using EST profiling analysis, genes likely to be involved in galactomannan biosynthesis were identified in addition to the known ManS and galactomannan galactosyl transferase genes. The second most highly expressed gene, called TfDUF246 in the previous report () but named TfMSR here (for ‘mannan synthesis-related’; Genbank accession number JX237834), encodes a protein that is likely to be involved in mannan biosynthesis. Here, we have used molecular, cellular, genetic and biochemical approaches to characterize TfMSR and its two Arabidopsis homologs, AtMSR1 and AtMSR2. The results provide evidence in support of our hypothesis that these proteins are involved in mannan biosynthesis.
nans, covering a wide range of Man/Gal ratios formed in vitro using the enzymes from the high-, medium- and low-galactose species. To allow comparison of probability-sets derived from galactomannans with different Man/Gal ratios all probability sets were scaled linearly, with Poo being ascribed the arbitrary value 1.0. When this was done it was observed that all probability sets from a single species were closely similar, yet quite different from those obtained from the other two species. This confirmed the correctness of the second-order Markov-chain model, and demonstrated clearly that the specificities of the galactomannan-forming transferase enzymes were different in the three species. The transfer specificities, defined mathematically in the probability sets, may each be considered to define a maximum Man/Gal ratio for a galactomannan synthesized according to the transfer rules thus defined. This is the Man/Gal ratio of the galactomannan formed when the highest of the four probabilities within a set is set to 1.0 and the other three are rescaled correspondingly in a linear fashion. When this was done for the fenugreek, guar and senna consensus probability sets, the maximum galactose-content predicted for each species was either identical with or only very slightly higher than that of the primary product of the biosynthetic machinery. This was remarkable, indicating not only that the distribution of substituents on the main chain of the galactomannan, but also the total amount of substitution is determined by the specificities of the membrane-bound transferase enzymes (Reid et al., 1995).
involved in galactomannan biosynthesis, ..
Based on the accumulation of mannose and on gene expression levels, eight CELLULOSE SYNTHASE-LIKEA genes (CSLA), which are highly likely to be related to the biosynthesis of bioactive mannan polysaccharides, were identified from the differentially expressed genes database.
The characterization of MSR protein demonstrates that biosynthesis of mannan polymers involves more proteins than ManS and galactomannan galactosyl transferase, and raises interesting questions such as how MSR protein affects ManS activity and what other proteins are involved in this process. Future studies aimed at providing answers to these questions will advance the understanding of mannan polysaccharide biosynthetic processes.
Biosynthesis of salep mannan - ScienceDirect
Two mechanisms are involved, the biosynthetic process, and a post-depositional modification. In fenugreek and guar, the Man/Gal ratios (1.1 and 1.6, respectively) are determined by the biosynthetic mechanism alone. On the other hand in senna, the Man/Gal ratio (3.3) is estabhshed by the controlled removal of galactose by a-galactosidase action from a primary biosynthetic product (Man/ Gal ratio = 2.3) during late seed development (Edwards et al., 1992). Post-depositional modification may be of much wider importance in the control of the structures of other cell wall polysaccharides than has been supposed hitherto. In the case of pectin, the removal of methyl ester groups by pectin methylesterase action is known to occur.
Effects of elicitors (mannan, β-1,3-glucan, and ancymidol) on the activity of several key enzymes participating in lignan biosynthesis were studied in Linum austriacum L. cell cultures. The activities of L-phenylalanine ammonia-lyase, polyphenoloxydase, tyrosine ammonia-lyase, soluble phenoloxidase, and membrane-bound and soluble oxidases were assayed. The elicitors under study affected various steps in the metabolic pathway of lignan biosynthesis. Elevated enzyme activity accompanied an elicitor-enhanced synthesis of podophyllotoxins and peltatins.
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Structure and biosynthesis of yeast cell wall mannan - …
If MSR proteins are directly involved in mannan biosynthesis, they should be localized to the Golgi apparatus where mannans and other matrix polysaccharides are synthesized. Using proteomic techniques, , ) and ) previously reported that AtMSR1 and AtMSR2 were localized to the Golgi apparatus. To examine the localization of TfMSR and confirm the localization of its two Arabidopsis homologs, a GFP-tagged version of each protein was transiently expressed together with a Golgi marker (ST-mRFP) () in tobacco leaves, and the localization was examined using live-cell confocal microscopy. As expected, GFP-tagged MSR proteins co-localized with the Golgi marker ST-mRFP in punctate structures (), indicating their localization in the Golgi. GFP-tagged MSR proteins were also found in some punctate structures where the ST-mRFP signal was not present or did not completely overlap with the GFP signal. Similar patterns showing incomplete overlap of signal were also found in a localization study for AtCSLA9 and AtCSLC4 (), and may indicate localization to the trans-Golgi network or secretory structures outside the Golgi proper.
is involved in mannan biosynthesis, ..
To determine whether AtMSR genes are involved in mannan biosynthesis, neutral monosaccharide composition analysis was performed using alcohol-insoluble residue (AIR) from the primary stems of WT and msr mutant plants (). The mannosyl levels decreased by approximately 40% in msr1-1 and msr1-2 single mutants, and further decreased to less than 50% of WT levels in the double mutants. However, no significant changes in the mannosyl levels were found in the three msr2 single mutants. Despite differences in fucosyl and arabinosyl levels found between msr1-1 and the WT, the results were not consistent in that the differences were not found in msr1-2 or the double mutants. In addition, the level of fucosyl residues was very low, and this low signal may contribute to the poor signal/noise ratio. We also analyzed the crystalline cellulose content in the stems of the mutants, and found no significant changes.
Biosynthesis of yeast mannan. Isolation of …
Because a method for fenugreek transformation has not been established, and genetic tools are not available, it is very difficult to perform molecular genetic analysis of MSR function in fenugreek. Therefore, we sought to identify Arabidopsis homologs so that the power of Arabidopsis molecular genetics could be used in functional characterization of this gene. The deduced amino acid sequence of TfMSR was used as a query to search against the Arabidopsis protein database () using BLASTP. This analysis yielded two homologs, At3g21190 and At1g51630, which were called AtMSR1 and AtMSR2, respectively. TfMSR shows 47 and 45% sequence identity, and 67 and 65% sequence similarity, to AtMSR1 and AtMSR2, respectively, and AtMSR1 shows 83% sequence identity and 91% sequence similarity to AtMSR2 (). The three proteins also have similar sizes (413, 422 and 423 amino acids) and were predicted to have a transmembrane domain at the N-terminus and a large conserved domain at the C-terminus (). Because AtMSR1 and AtMSR2 are homologs of TfMSR, we postulated that they may also be involved in mannan biosynthesis.
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