J. Richard Dickinson

Purification and characterization of hydroxycinnamate decarboxylase from Brettanomyces anomalus

Abstract The yeast, Brettanomyces anomalus , produces a hydroxycinnamic acid decarboxylase which is active toward ferulic acid, p -coumaric acid, and caffeic acid. The enzyme transforms these hydroxycinnamic acids to hydroxystyrenes by the removal of the carboxyl group from the C3 side chain. We have purified this enzyme 235-fold from B. anomalus NCYC 615 using Mono Q ion exchange, Phenyl Superose, and Superose 12 column chromatography. Enzyme activity was found to be optimal at 40°C and pH 6.0 and was enhanced by EDTA, Mg 2+ , and Cr 3+ . Fe 3+ , Ag + , and SDS completely inhibited the activity. Kinetic studies indicated a K m of 1.15 m m and a V max of 13,494 nmol min −1 mg −1 for ferulic acid and a K m of 1.55 mM and a V max of 22,256 nmol min −1 mg −1 for p -coumaric acid. Using gel filtration, an apparent molecular mass of 39.8 kDa was estimated. The decarboxylase was inactive toward both o - and m -coumaric acid and toward cinnamic acid, indicating that the para -hydroxy group is essential for activity.

A study of branched‐chain amino acid aminotransferase and isolation of mutations affecting the catabolism of branched‐chain amino acids in Saccharomyces cerevisiae

The specific activity of branched‐chain amino acid aminotransferase was highest when S. cerevisiae was grown in minimal medium containing a branched‐chain amino acid as nitrogen source. Growth in complex media with glycerol or ethanol gave moderately high levels, whereas with glucose and fructose the specific activity was very low. Mutagenesis defined three genes (BAA1 to BAA3) required for branched‐chain amino acid catabolism. The baal mutation reduced the specific activity of the aminotransferase, the stationary phase density in YEPD and caused gross morphological disturbance. Branched‐chain amino acid aminotransferase is essential for sporulation.

A simple method for the direct extraction of plasmid DNA from yeast

A rapid and simple method for the small scale isolation of shuttle plasmid DNA from Saccharomyces cerevisiae is described. It uses glass beads to break cells and reagents which are also used in bacterial mini-preps to yield plasmid DNA without chromosomal contamination in sufficient quantities to enable direct visualisation on agarose gels.

Increased activity of a model heterologous protein in Saccharomyces cerevisiae strains with reduced vacuolar proteinases

Strains of Saccharomyces cerevisiae with reduced activity of the four major vacuolar proteinases were constructed and used as an expression system for a model heterologous gene product (β-galactosidase from Escherichia coli). The vacuolar proteinases were inactivated by mutation within the structural genes encoding proteinase A (PRA1), proteinase B (PRB1), carboxypeptidase Y (PRC1) and carboxypeptidase S (CPS1). Strains were constructed with mutations in one or more of these structural genes. Having constructed the strains, the E. coli β-galactosidase (lacZ) gene was introduced by transformation. Batch cultures of each strain were grown and the activity of β-galactosidase measured. An assessment of the effect of the loss of specific proteinases on the heterologous gene product was then made. The results indicated that strains with reduced vacuolar proteinase activity showed as much as 173% higher β-galactosidase activity than a strain with wild-type proteinase activity carrying the lacZ gene. The most productive strains of all were those with reduced carboxypeptidase activity and/or reduced proteinase A activity. At first sight the inclusion of a pra1 mutation and/or the pra1 and cps1 mutations would appear wortwhile for significantly enhanced expression of a heterologous gene product in yeast. However this conclusion is too simplistic: each heterologous protein will require a host specifically “tailored” to ensure optimum expression.

Combined 13C NMR- and mass-spectrometry for non-invasive monitoring of metabolism

A method for the long-term incubation of cell suspensions at measured dissolved gas concentrations during acquisition of NMR spectra is described: a mass spectrometer probe in the cell suspension provides continuous monitoring of gases. Its usefulness is shown for Trichomonas vaginalis where fermentation products from glucose in 72 μM O2 are different from those under strictly anaerobic conditions.

The activity of a model heterologous protein in pep4-3 mutants of Saccharomyces cerevisiae

SummaryThe bacterial lacZ gene was introduced into two sibling strains of Saccharomyces cerevisiae, one a wild-type strain with normal proteinase activity and the other a pep4-3 mutant strain. The pep4-3 mutation resulted in 90% reduced activity of the four major vacuolar proteinases. By comparing the activity of the lacZ gene product (β-galactosidase) in both strains the degradative effect of the major vacuolar proteinases on a heterologous protein was estimated. The mutant strain with reduced proteinase activity had higher β-galactosidase activity under all the test conditions. In the most productive case the pep4-3 mutant had 55% higher β-galactosidase activity than the wild-type. Batch cultures of the two strains were evaluated for growth characteristics. The strain with reduced proteinase activity grew to higher optical densities than the wild-type. Upon further examination it was found that not only were the optical densities of pep4-3 cultures greater but the cell numbers were much greater than expected due to the smaller size of pep4-3 cells. It is concluded that the strain lacking vacuolar proteinases maintained increased levels of β-galactosidase and is physiologically as healthy as the wild-type.

Chapter 1 Control of the cell cycle

Publisher Summary The process of mitosis is, as viewed by phase contrast microscopy, an extremely dynamic one that involves rapid reorganization of the cytoskeleton and the establishment of two opposing cellular poles. These then act as foci for orientation of replicated chromosomes. As yet, basic understanding of the mechanism and control of this massive spatial reorganization is in its infancy. Therefore, current insights into the pathology of defective cell division are still severely limited at the level of cell ultra structure. Programmed cell death occupies a special place midway between the cell cycle, development, and aging. In mammalian cells, it appears that special genes are reserved to ensure termination of a specific cell at a pre-ordained time. There is a growing interest in identifying such genes, some of which have been identified in simpler eukaryotes, such as bakers yeast, which also shows aging.