Respiratory mutation and galactose metabolism in yeast Saccharomyces cerevisiae.

Restriction in growth on galactose as unique source of energy due to respiratory deficiency resulting from mutation in a gene gal probably different from gal 3 is described.     

Sequence and structure of the yeast galactose transporter.

The previously cloned GAL2 gene of the Saccharomyces cerevisiae galactose transporter has been sequenced. The nucleotide sequence predicts a protein with 574 amino acids (Mr, 63,789). Hydropathy plots suggest that there are 12 membrane-spanning segments. The galactose transporter shows both sequence and structural homology with a superfamily of sugar transporters which includes the human HepG2-erythrocyte and fetal muscle glucose transporters, the rat brain and liver glucose transporters, the Escherichia coli xylose and arabinose permeases, and the S. cerevisiae glucose, maltose, and galactose transporters. Sequence and structural motifs at the N-terminal and C-terminal regions of the proteins support the view that the genes of this superfamily arose by duplication of a common ancestral gene. In addition to the sequence homology and the presence of the 12 membrane-spanning segments, the members of the superfamily show characteristic lengths and distributions of the charged, hydrophilic connecting loops. There is indirect evidence that the transporter is an N-glycoprotein. However, its only N-glycosylation site occurs in a charged, hydrophilic segment. This could mean that this segment is part of a hydrophilic channel in the membrane. The transporter has a substrate site for the cyclic AMP-dependent protein kinase which may be a target of catabolite inactivation. The transporter lacks a strong sequence enriched for proline (P), glutamate (E), aspartate, serine (S), and threonine (T) and flanked by basic amino acids (PEST sequence) even though it has a short half-life. Mechanisms for converting the poor PEST to a possible PEST sequence are considered. Like the other members of the superfamily, the galactose transporter lacks a signal sequence.     

The genomics of adaptation in yeast

A recent study has combined methods of experimental evolution and DNA microarray technology to examine evolved changes in gene expression in yeast, providing intriguing insights into the genetics of adaptation and functional genomics, and pointing to future uses of microarray technology in evolutionary genetics.     

The role of recombination and RAD52 in mutation of chromosomal DNA transformed into yeast.

While transformation is a prominent tool for genetic analysis and genome manipulation in many organisms, transforming DNA has often been found to be unstable relative to established molecules. We determined the potential for transformation-associated mutations in a 360 kb yeast chromosome III composed primarily of unique DNA. Wild-type and rad52 Saccharomyces cerevisiae strains were transformed with either a homologous chromosome III or a diverged chromosome III from S. carlsbergensis. The host strain chromosome III had a conditional centromere allowing it to be lost on galactose medium so that recessive mutations in the transformed chromosome could be identified. Following transformation of a RAD+ strain with the homologous chromosome, there were frequent changes in the incoming chromosome, including large deletions and mutations that do not lead to detectable changes in chromosome size. Based on results with the diverged chromosome, interchromosomal recombinational interactions were the source of many of the changes. Even though rad52 exhibits elevated mitotic mutation rates, the percentage of transformed diverged chromosomes incapable of substituting for the resident chromosome was not increased in rad52 compared to the wild-type strain, indicating that the mutator phenotype does not extend to transforming chromosomal DNA. Based on these results and our previous observation that the incidence of large mutations is reduced during the cloning of mammalian DNA into a rad52 as compared to a RAD+ strain, a rad52 host is well-suited for cloning DNA segments in which gene function must be maintained.     

The effect of sex on adaptation to high temperature in heterozygous and homozygous yeast.

Most explanations for the evolutionary maintenance of sex depend on the assumption that sex produces variation by recombining parental haplotypes in the offspring. Therefore, meiosis is expected to be useful only in heterozygotes. We tested this assumption by competing sexual strains of yeast against constitutive asexuals in a hot (37 degrees C) culture for 500 generations, in either heterozygous or homozygous genetic backgrounds. We found that there was an initial cost of sex for all the sexual strains, which was indicated by a sharp increase in the proportion of asexuals after the induction of sex. The cost was larger in the heterozygotes than in the homozygotes, probably because of recombinational load. However, in two of the three heterozygote backgrounds, after the initial success of the asexuals, the remaining sexuals eventually drove them out of the population. These two heterozygotes also suffered the largest initial cost of sex. In the other heterozygote and in the three homozygote backgrounds it appeared to be a matter of chance whether sexuals or asexuals won. The average relative fitness increased in all the strains, but the increase was largest in the two strains that showed both the clearest advantage and the largest cost of sex. We conclude that these results are consistent with the traditional view that sex has a short-term cost but a long-term benefit.     

An investigation of the role of the copper in galactose oxidase.

Galactose oxidase is a metalloenzyme containing a single copper atom per molecule. The mechanism of action of galactose oxidase is studied in this paper by investigating substrate specificity and activation by peroxidase, and probing the copper site by electron spin resonance (ESR) spectroscopy. Line-shape simulation of ESR spectra are also reported and a comparison is made between observed and simulated spectra for galactose oxidase. A comparison is also reported for the enzyme from various commercial sources and enzyme isolated from a fungus in this laboratory. The results of this investigation suggest that the copper is in an environment of four in-plane nitrogens with axial symmetry.     

The role of phytosterols in plant adaptation to temperature

Membranes of composition approaching those found in “rafts” of plants, fungi and mammals were investigated by means of solidstate ²H-NMR, using deuterated dipalmitoyl-phosphatidylcholine (²H-DPPC) as a reporter. The dynamics of such membranes was determined through measuring of membrane ordering or disordering properties. The presence of the liquid-ordered, lo, phase, as an indicator of rigid sterol-sphingolipid domains, was detected in all cases. Of great interest, the dynamics of mixtures mimicking rafts in plants showed the lesser temperature sensitivity to thermal shocks. The presence of an additional ethyl group branched on the alkyl chain of major plant sterols (sitosterol and stigmasterol) is proposed as reinforcing the membrane cohesion. The fine tuning of the sterol structure thus appears to be the evolution response for plant adaptation to large temperature variations.Key words: sitosterol, stigmasterol and glucosylcerebrosides, regulation of membrane dynamics, membrane rafts, deuterium NMRIt is widely recognized that lipids play multiple roles that either individually or collectively influence cell processes. Glycerolipids and sphingolipids through charge and structure are involved in DNA replication, protein translocation, cell recognition, signalling pathways, energetic, signal transduction, and cell trafficking. Together with diacylglycerols their collective properties modulate lipid polymorphism, through phase transitions (lamellar, hexagonal, cubic, micelles), which are involved in enzyme conformational changes, cell division, cell fusion, and apoptosis.¹Sterols, the third lipid class, also regulate biological processes and sustain the domain structure of cell membranes where they are considered as membrane reinforcers.^2,3 While cholesterol (CHO) is the major sterol of vertebrates, ergosterol plays a key role in fungi. Plants usually possess more complex sterol compositions. Stigmasterol (STI) and sitosterol (SIT), two 24-ethyl sterols, are major constituents of the sterol profiles of plant species. They are involved in the embryonic growth of plants.^4,5 Sterols are critical for the formation of liquid-ordered (lo) lipid domains (lipid rafts) that are supposed to play an important role in fundamental biological processes like signal transduction, cellular sorting, cytoskeleton reorganization and infectious diseases.^6,7 In plants, specialized lipid domains are involved in the polarized growth of pollen tube and root hair⁸ and the asymmetric growth of plant cells is in general due to the asymmetric distribution of membrane components.We recently documented the effect of sitosterol and stigmasterol, two major plant sterols, on the structure and dynamics of membranes whose composition is representative of domains (rafts) in plants.⁹ Liposomes of phytosterols associated with glucosylcerebroside (GC) and with deuterium-labelled dipalmitoylphosphatidylcholine (²H-DPPC) were analysed with deuterium solid state nuclear magnetic resonance (²H-NMR). For comparison, membrane systems representative of raft composition in fungi and mammals were also investigated. ²H-NMR is known to be the best non-invasive technique to analyse membrane dynamics¹⁰ because it is non-destructive and because replacement of DPPC protons with their deuterium isotope brings very little membrane perturbation.^11,12 Acyl chain deuteration affords analysis of both structure and dynamics of the hydrophobic membrane interior. Spectra such as that shown in Figure 1 insert, allow detection of the lo phase, characteristic of a membrane state half-way between solid-ordered (so) and liquid-disordered (ld) states. The so state, also called “gel”, is found at low temperatures (below 35°C), when membranes are essentially composed of sphingomyelins (SM)¹³ or GC (Fig. 1). This membrane state allows little biological function because in forbids membrane trafficking due to its very rigid state (order parameter close to 1). In turn, the ld or “fluid” state is found at high temperatures, in the absence of SM, GC and sterols (low order parameter). At the opposite such a high membrane dynamics may lead to excessive membrane passages. Following with ²H-NMR the temperature behaviour of membrane systems containing GC and plant sterols, we found that the so-ld, order-disorder, transition was totally abolished: SIT and STI fluidized the so state and ordered the ld state to produce the lo state where membrane fluctuations vary smoothly with temperature (Fig. 1). This effect was already documented with CHO in mammals^14–16 but on a much narrower temperature range. The case of the fungus system was found in between that of plants and mammals.Open in a separate windowFigure 1Regulation of temperature-driven membrane dynamics by plant sterols. Central panel: first spectral moment (left y-axis) or order parameter (right y-axis) as a function of temperature; solid line: ²H-DPPC with glucosylcerebroside; open circles: plus stigmasterol; filled circles: plus sitosterol. Insert: ²H-NMR spectrum typical of a liquid-ordered, lo, state. Left panel: schematics of solid-ordered, so (gel), and liquid-disordered, ld (fluid), membrane states. Right panel: schematics of the lo (raft) membrane state together with the structures of cholesterol and sitosterol. Adapted from reference ⁹.Summarizing, it appears that plant membranes of “raft” composition are less sensitive to temperature variations than those of animals. This suggests that cell membrane components like sitosterol, stigmasterol and glucosylcere-brosides, which are typical of plants, are produced in order to extend the temperature range in which membrane-associated biological processes can take place. This observation is well in accordance with the fact that plants have to face higher temperature variations than animals, which usually can either regulate their body temperature or change their location in order to avoid extreme heat or coldness.Compared to cholesterol, the two phytosterols possess additional ethyl groups branched on C-24 (Fig. 1). We proposed that the presence of an additional ethyl group may reinforce the attractive van der Waals interactions leading to more membrane cohesion and therefore less temperature sensitivity. Our results also suggest that domains of smaller size would be promoted in the presence of phytosterols and especially with sitosterol. Such domains may be viewed as dynamic, with sterols laterally exchanging at the microsecond time scale.¹⁴ In plant cells, enzymes transfer alkyl groups to the C-24 of sterols. If we suppose that the relative activities of the different branches of the plant sterol biosynthesis are regulated, the concentrations of major sterols in plants, like sitosterol, stigmasterol, and cholesterol could be controlled.^4,17 This shows the importance of equilibrated sterol concentrations for plant growth and development. Sterols have been historically considered as membrane reinforcers because they bring order to membranes.^2,3Our works^{9,15,16,18,19} show that they could better be named as “membrane dynamics regulators”, by maintaining the membrane in a state of microfluidity suitable for cell function on large temperature scales. It thus appears that a fine tuning of the sterol structure, i.e., the presence of branched ethyl groups in plant sterols increasing membrane cohesion through formation of smaller membrane domains, may be the evolution response for plant adaptation to large temperature variations.