Saccharomyces cerevisiae a cells secrete an extracellular protein, called "barrier" activity, that acts as an antagonist of a factor, the peptide mating pheromone produced by mating-type a cells. We report here the DNA sequence of BAR], the structural gene for barrier activity. The deduced primary translation product of 587 amino acids has a putative signal peptide, nine potential asparagine-linked glycosylation sites, and marked sequence similarity of the first two-thirds of the protein with pepsin-like proteases. Barrier activity was abolished by in vitro mutation of an aspartic acid predicted from this sequence homology to be in the active site. Therefore, barrier protein is probably a protease that cleaves a factor. The sequence similarity suggests that the first two-thirds of the barrier protein is organized into two distinct structural domains like those of the pepsin-like proteases. However, the BAR) gene product has a third carboxyl-terminal domain of unknown function; deletion of at least 166 of the 191 amino acids of this region has no significant effect on barrier activity.

The time course of synthesis and breakdown of various macromolecules has been compared for sporulating ( a /α) and nonsporulating ( a/a and α/α) yeast cells transferred to potassium acetate sporulation medium. Both types of cells incorporate label into ribonucleic acid and protein. The gel electrophoresis patterns of proteins synthesized in sporulation medium are identical for sporulating and nonsporulating diploids; both are different from electropherograms of vegetative cells. Sporulating and nonsporulating strains differ with respect to deoxyribonucleic acid synthesis; no deoxyribonucleic acid is synthesized in the latter case, whereas the deoxyribonucleic acid complement is doubled in the former. Glycogen breakdown occurs only in sporulating strains. Breakdown of preexisting vegetative ribonucleic acid and protein molecules occurs much more extensively in sporulating than in nonsporulating cells. A timetable of these data is presented.

The purpose of this study was to characterize changes in gene expression in the brain of a seasonal hibernator, the goldenmantled ground squirrel, Spermophilus lateralis, during the hibernation season. Very little information is available on molecular changes that correlate with hibernation state, and what has been done focused mainly on seasonal changes in peripheral tissues. We produced over 4000 reverse transcription-PCR products from euthermic and hibernating brain and compared them using differential display. Twenty-nine of the most promising were examined by Northern analysis. Although some small differences were observed across hibernation states, none of the 29 had significant changes. However, a more direct approach, investigating expression of putative hibernationresponsive genes by Northern analysis, revealed an increase in expression of transcription factors c-fos, junB, and c-Jun, but not junD, commencing during late torpor and peaking during the arousal phase of individual hibernation bouts. In contrast, prostaglandin D2 synthase declined during late torpor and arousal but returned to a high level on return to euthermia. Other genes that have putative roles in mammalian sleep or specific brain functions, including somatostatin, enkephalin, growth-associated protein 43, glutamate acid decarboxylases 65/67, histidine decarboxylase, and a sleep-related transcript SD464 did not change significantly during individual hibernation bouts. We also observed no decline in total RNA or total mRNA during torpor; such a decline had been previously hypothesized. Therefore, it appears that the dramatic changes in body temperature and other physiological variables that accompany hibernation involve only modest reprogramming of gene expression or steady-state mRNA levels.

Proteins secreted by the yeast Saccharomyces cerevisiae are usually modified by the addition at asparagine-linked glycosylation sites of large heterogeneous mannan units that are highly immunogenic. Secreted proteins from mnnl mnn9 mutant strains, in contrast, have homogeneous Man1*GlcNAc2 oligosaccharides that lack the immunogenic al,3-mannose linkages. We have cloned and sequenced the MNN9 and MNNI genes, both of which encode proteins with the characteristics of type I membrane proteins. Mnn9p is a membrane-associated protein with unknown function that is required for the addition ofthe long al,6-mannose backbone of the complex mannan, whereas Mnnip is most likely the al,3-mannosyltransferase located in the Golgi apparatus.The addition of core oligosaccharide to Asn-Xaa-Ser/Thr (N-linked) glycosylation sites in secreted proteins appears to follow similar pathways in the yeast Saccharomyces cerevisiae and mammalian cells (1). In both, lipid-linked oligosaccharide containing N-acetylglucosamine, mannose, and glucose (Glc3Man9GlcNAc2) is transferred to the asparagine residues in secreted proteins in the lumen of the endoplasmic reticulum (ER). Studies with mammalian proteins expressed in yeast have demonstrated that yeast generally recognizes the same N-linked glycosylation sites as mammalian cells (2-4). The oligosaccharide is then trimmed to the core oligosaccharide Man8GlcNAc2 before transport of the glycoprotein to the Golgi compartment, where additional modifications occur.In the mammalian Golgi apparatus, several mannoses in the core oligosaccharide are usually trimmed before the addition of a limited number of other sugars (e.g., galactose, fucose, and sialic acid) in the "complex-type" carbohydrate chains; "high-mannose" glycosylation results from the absence of or incomplete mannose trimming. In contrast, yeast can extend the core oligosaccharide with a long al,6-linked mannose chain, which is then further modified by the addition of al,2-and al,3-mannose side chains. This hyperglycosylation significantly contributes to the high molecular mass and heterogeneity ofmost secreted yeast proteins (5-7), although not all core oligosaccharides are necessarily hyperglycosylated (8, 9).Extensive genetic and biochemical characterization of the mannans on S. cerevisiae cell wall mannoproteins (analogous to the mannan on secreted protein) by Ballou and coworkers (reviewed in refs. 10 and 11) has generated mnn mutants that are defective in mannan biosynthesis. Four of these (mnn7, mnn8, mnn9, and mnnJO) produce mannoproteins that are significantly underglycosylated relative to wild-type strains or other mnn mutants. These four mutants were also the first in which the mnn mutation had a deleterious effect on such characteristics as cell growth, osmotic sensitivity, and ascospore viability. Subsequent work with the mnn9 mutant demonstrated that one al,6-linked mannose is added to the core oligosaccharide but that further extension of the al,6 backbone is blocked, although limited al,2 and al,3 mannose moieties ...

Several lines of investigation suggest that the serotonergic system may be involved in the pathogenesis of migraine. In particular, drugs which block 5-HT2 receptor subtypes appear to be effective migraine prophylactic agents. Therefore, chromosomal DNA regions overlapping the 5-HT2A (13q14-q22) and 5-HT2C(Xq22-25) receptor loci were analyzed for possible linkage to the clinical diagnosis of migraine. No evidence for linkage to either chromosomal region was found, although a small subset of migrainous families showed positive likelihood of odds (LOD) scores. However, a homogeneity (HOMOG) analysis provided no statistical evidence for locus heterogeneity. The coding region of the 5-HT2A and 5-HT2C receptor genes was also analyzed in migraine patients and unaffected controls using polmerase chain reaction and direct sequencing. No mutations were found in the deduced amino acid sequence of either receptor in the sample of migraineurs tested. These results indicate that DNA-based mutations in the 5-HT2A and 5-HT2C receptors are not generally involved in the pathogenesis of migraine.