An <i>in vitro</i> assay for (1 → 6)‐β‐<scp>D</scp>‐glucan synthesis in <i>Saccharomyces cerevisiae</i>
Bibliographic record
Abstract
(1 --> 6)-beta-D-glucan is a key cell wall component of Saccharomyces cerevisiae and Candida albicans. Many genes are known to affect the levels or structure of this glucan, but their roles and a molecular description of the synthesis of (1 --> 6)-beta-D-glucan remain to be established and a method to measure (1 --> 6)-beta-D-glucan synthase activity in vitro would provide an enabling tool. Here, conditions for the detection of in vitro synthesis of this polymer are described. Crude membrane preparations from S. cerevisiae were isolated, and incubated in the presence of UDP-glucose and GTP. With anti-(1 --> 6)-beta-D-glucan-specific antibodies, a time-dependent increase in the amount of this glucan was demonstrated in a dot-blot assay, or through an inhibition enzyme immunoassay. Antibody specificity was validated by competition experiments using pustulan, a (1 --> 6)-beta-D-glucan, laminarin, a (1 --> 3)-beta-D-glucan, yeast mannan and glycogen. The identity of the reaction product was also demonstrated by its sensitivity to a recombinant (1 --> 6)-beta-D-glucanase. Extracts from mutants in 10 genes with a wide range of altered cell wall (1 --> 6)-beta-D-glucan levels were assayed for in vitro synthesis of the polymer. A strong correlation of in vitro synthase activity with in vivo glucan levels was found, providing genetic support for the specificity of the assay. The basis for the GTP-dependence of the synthase reaction was studied. Extracts from rho2, rho3, rho4 and rho5 null mutants had wild-type in vitro activity. In contrast, Rho1p overproduction led to increased in vitro synthesis, implicating Rho1p in the regulation of (1 --> 6)-beta-D-glucan synthesis.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.002 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".