Galactose Transport in Saccharomyces cerevisiae III. Characteristics of Galactose Uptake in Transferaseless Cells: Evidence Against Transport-Associated Phosphorylation

The characteristics of the inducible galactose transport system in bakers' yeast were studied in uridine diphosphate, galactose-1-phosphate uridylyl-transferaseless cells. Transferaseless cells transport galactose at the same initial rate as wild-type cells and accumulate a mixture of free galactose and galactose-1-phosphate. The addition of ¹⁴C-labeled galactose to cells preloaded with unlabeled galactose and galactose-1-phosphate results in a higher rate of labeling of the free-sugar pool than of the galactose-1-phosphate pool. These results support other evidence that galactose uptake in bakers' yeast is a carrier-mediated, facilitated diffusion and that phosphorylation is an intracellular event after uptake of the free sugar.     

Predation risk and moonlight avoidance in nocturnal seabirds

Unlike most seabird families, the vast majority of small petrel species are nocturnal on their breeding grounds. Further, they reduce markedly their activity when the light level increases. Moonlight avoidance might be a consequence of reduction in foraging profitability, as bioluminescent prey do not come to the sea surface on bright nights. Alternatively, petrels may avoid colonies during moonlit nights because of increased predation risk. We studied predation on petrels by Brown Skuas Catharacta antarctica lönnbergi at Kerguelen, and the influence of moonlight on behaviour of both skuas and petrels, to test the 'predation risk' hypothesis. On the study area, Brown Skuas hunt at night and prey heavily upon the Blue Petrel Halobaena caerulea and the Thin-billed Prion Pachyptila belcheri . Predation risk was higher on moonlit nights, as skuas caught more prey, and particularly more Blue Petrels when the light level increased. Nightly intakes of Blue Petrel and Thin-billed Prion by skuas was related to colony attendance of non-breeders rather than that of breeders. Biometry of prey also suggested that skuas caught a higher proportion of non-breeding birds than was present at the colonies. Predation risk was thus greater in non-breeders and on moonlit nights. Colony attendance by non-breeding Blue Petrels and Thin-billed Prions was also reduced during moonlit nights. Vocal activity, which is mainly by non-breeders, was also drastically reduced when the light level increased in the species suffering the highest predation rate. Our results supported the 'predation risk' hypothesis, although the 'foraging efficiency' and the 'predation risk' hypotheses are not mutually exclusive: the former might explain the moonlight avoidance behaviour of breeding, and the latter that of non-breeding individuals.     

Polypyrimidine Tract Binding Protein Prevents Activity of an Intronic Regulatory Element That Promotes Usage of a Composite 3��-Terminal Exon

Alternative splicing of 3′-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3′-untranslated regions that regulate their fate and their expression. The Xenopus α-tropomyosin pre-mRNA possesses a composite internal/3′-terminal exon (exon 9A9′) that is differentially processed depending on the embryonic tissue. Exon 9A9′ is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3′-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9′ as a 3′-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9′ in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9′ as a 3′-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3′-end processing of the α-tropomyosin pre-mRNA.     

A familial mutation in the testis-determining gene SRY shared by both sexes

A familial mutation in SRY, the gene coding for the testis-determining factor TDF, was identified in an XY female with gonadal dysgenesis, her father, her two brothers and her uncle. The mutation consists of a T to C transition in the region of the SRY gene coding for a protein motif known as the high mobility group (HMG) box, a protein domain known to confer DNA-binding specificity on the SRY protein. This point mutation results in the substitution, at amino acid position 109, of a serine residue for phenylalanine, a conserved aromatic residue in almost all HMG box motifs known. This F109S mutation was not found in 176 male controls. When recombinant wildtype SRY and SRY^F109S mutant protein were tested in vitro for binding to the target site AAC AAAG, no differences in DNA-binding activity were observed. These results imply that the F109S mutation either is a rare neutral sequence variant, or produces an SRY protein with slightly altered in vivo activity, the resulting sex phenotype depending on the genetic back-ground or environmental factors.This paper is dedicated by G. S. to Professor Ulrich Wolf on the occasion of his 60th birthday     

Cysteine control over glutathione homeostasis in Chinese hamster fibroblasts overexpressing a gamma-glutamylcysteine synthetase activity.

Gamma-glutamylcysteine synthetase (GCS) catalyses the first step of glutathione (GSH) biosynthesis and is considered to be the rate-limiting step of this pathway. In several experimental systems, GCS overexpression has been associated with GSH pool expansion and drug resistance. In this report, we describe a mutant line of Chinese hamster fibroblasts that overexpress this activity by 4-5 times, due to the amplification of the gene encoding the catalytic subunit of GCS. These mutant cells contained a wild-type steady-state level of GSH and, after depletion, synthesized GSH at the same rate as wild-type cells because their rate of endogenous production of cysteine was limiting. An exogenous supply of cysteine expanded the pool of GSH in mutant cells by 80% but did not increase that of wild-type cells, and, in GSH-depleted cells, increased the rate of GSH biosynthesis by eight and 35-times in wild-type and mutant cells, respectively. These experiments indicated that GCS overexpression had no consequence on the metabolism of GSH, unless a supply of cysteine was provided. Mutant cells were not resistant to cisplatin or nitrogen mustard.     

Mechanistic studies on aromatase and related C---C bond cleaving P-450 enzymes

Some P-450 systems, notably aromatase and 14-demethylase catalyse not only the hydroxylate reaction but also the oxidation of an alcohol into a carbonyl compound as well as a C---C bond cleavage process. All these reactions occur at the same active site. A somewhat analogous situation is noted with 17-hydroxylase-17,20-lyase that participates in hydroxylation as well as C---C bond cleavage process. The C---C bond cleavage reactions catalysed by the above enzymes conform to the general equation:

Full-size image

It is argued that all three types of reaction catalyzed by these enzymes may be viewed as variations on a common theme. In P-450 dependent hydroxylation the initially formed Fe^III---O---O^. species is converted into Fe^III---O---OH and the heterolysis of the oxygen—oxygen bond of the latter then gives the oxo-derivative for which a number of canonical structures are possible; for example Fe^V = O ↔ (+^.)Fe^IV = O ↔ Fe^IV---O^.. One of these, Fe^IV---O^. behaves like an alkoxyl radical and participates in hydrogen abstraction from C---H bond to produce Fe^IV---OH and carbon radical. The latter is then quenched by the delivery of hydroxyl radical from Fe^IV---OH. The latter species may thus be regarded as a carrier of hydroxyl radical. We have proposed that the C---C bond cleavage reaction occurs through the participation of the Fe^III---O---OH species that is trapped by the electrophilic property of the carbonyl compound giving a peroxide adduct that fragments to produce an acyl—carbon cleavage. Scientific developments leading up to this conclusion are considered. In the first author's views,

“The study of mechanisms is not a scientific but a cultural activity. Mechanisms do not aim at an absolute truth but are intended to be a “running” commentary on the status of knowledge in a field. As the structural knowledge in a field advances Mechanisms evolve to take note of the new findings. Just as a constructive “running” commentary provides the stimulus for higher standards of performance, so Mechanisms call for better and firmer structural information from their practitioners”.