Genetic dissection of the signaling domain of a mammalian steroid receptor in yeast.

The mechanism of signal transduction by steroid receptor proteins is complex and not yet understood. We describe here a facile genetic strategy for dissection of the rat glucocorticoid receptor "signaling domain," a region of the protein that binds and transduces the hormonal signal. We found that the characteristics of signal transduction by the receptor expressed in yeast were similar to those of endogenous receptors in mammalian cells. Interestingly, the rank order of particular ligands differed between species with respect to receptor binding and biological efficacy. This suggests that factors in addition to the receptor alone must determine or influence ligand efficacy in vivo. To obtain a collection of receptors with distinct defects in signal transduction, we screened in yeast an extensive series of random point mutations introduced in that region in vitro. Three phenotypic classes were obtained: one group failed to bind hormone, a second displayed altered ligand specificity, and a third bound hormone but lacked regulatory activity. Our results demonstrate that analysis of glucocorticoid receptor action in yeast provides a general approach for analyzing the mechanism of signaling by the nuclear receptor family and may facilitate identification of non-receptor factors that participate in this process.     

HNO induces DNA deletions in the yeast S. cerevisiae

HNO is genotoxic but its mechanism is not well understood. There are many possible mechanisms by which HNO can attack DNA. Since HNO is electrophilic, it may react with exocyclic amine groups on DNA bases and through a series of subsequent reactions form a deaminated product. Alternatively, HNO may induce radical chemistry through O(2)-dependent (or possibly O(2)-independent) chemistry. In cell free systems, experiments have shown that HNO does react with DNA, resulting in base oxidation and strand cleavage. In this study, we used a whole-cell system in the yeast Saccharomyces cerevisiae to study the mechanism of HNO induced DNA damage with Angeli's salt as HNO donor. The yeast DEL assay provided a measure of intrachromosomal recombination leading to DNA deletions. We also examined interchromosomal recombination leading to genomic rearrangements and used the canavanine (CAN) assay to study induction of forward point mutations. HNO was a potent inducer of DNA deletions and recombination but it was negative for induction of point mutations. This suggests that HNO causes DNA strand breaks rather than base damage. Genotoxicity was observed under aerobic and anaerobic conditions and NAC protected against HNO induced DNA deletions. Since HNO is genotoxic under anaerobic conditions, NAC probably protected against radicals generated by HNO independent of oxygen.     

Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins

The ras genes, which were first identified by their presence in RNA tumor viruses and which belong to a highly conserved gene family in vertebrates, have two close homologs in yeast, detectable by Southern blotting. We have cloned both genes (RAS1 and RAS2) from plasmid libraries and determined the complete nucleotide sequence of their coding regions. They encode proteins with nearly 90% homology to the first 80 positions of the mammalian ras proteins, and nearly 50% homology to the next 80 amino acids. Yeast RAS1 and RAS2 proteins are more homologous to each other, with about 90% homology for the first 180 positions. After this, at nearly the same position that the mammalian ras proteins begin to diverge from each other, the two yeast ras proteins diverge radically. The yeast ras proteins, like the proteins encoded by the mammalian genes, terminate with the sequence cysAAX, where A is an aliphatic amino acid. Thus the yeast ras proteins have the same overall structure and interrelationship as the family of mammalian ras proteins. The domains of divergence may correspond to functional domains of the ras proteins. Monoclonal antibody directed against mammalian ras proteins immunoprecipitates protein in yeast cells containing high copy numbers of the yeast RAS2 gene.     

Site-specific AT-cluster insertions in the mitochondrial 15S rRNA gene of the yeast S. cerevisiae.

By comparing the mitochondrial 15S rRNA sequences of four wildtype yeast strains together with their respective secondary structures, with those of the 16S-like ribosomal RNA from other organisms we detected two optional and two invariant AT-clusters. The origin of these clusters is discussed with respect to their roles as possible mobile elements.     

Brave little yeast, please guide us to thebes: sphingolipid function in S. cerevisiae

Sphingolipids typically cover the exoplasmic leaflet of the plasma membrane of eukaryotic cells. They differ from the more abundant glycerophospholipids in that they contain ceramide instead of diacylglycerol as a hydrophobic anchor. Why did nature choose to invent this complex class of lipids, and why do eukaryotic cells follow elaborate remodelling pathways in order to generate dozens to hundreds of different molecular species of sphingolipid, depending on cell type? Yeast may, once again, serve as a model to dissect sphingolipid function at various levels. Almost the complete pathway for sphingolipid synthesis in yeast has been uncovered during the past two decades. More recently, key enzymes in sphingolipid degradation and signalling have been identified. Together with a wealth of genetic data obtained from the characterization of various suppressor mutants, this information now allows for an unprecedented analysis of sphingolipid function in this organism. This overview summarizes recent data on sphingolipid function in cell signalling, their role in the heat-stress response and Ca(2+) homeostasis, and addresses their function in transport of glycosylphosphatidylinositol-anchored proteins.     

RNA synthesis and control of cell division in the yeast S. cerevisiae.

Cells of the yeast Saccharomyces cerevisiae rapidly accumulated in the G1 phase of the cell cycle when exposed to the chelating agents o-phenanthroline (OP) or 8-hydroxyquinoline (HQ). Zinc salts fully reversed the growth-inhibitory effect of both OP and HQ. Cells treated with these chelating agents showed limited RNA accumulation and little RNA degradation. Rates of RNA synthesis were drastically reduced by low concentrations of these compounds. Whereas rates of protein synthesis were essentially unaffected. Rates of synthesis of mRNA and tRNA were less affected than were rates of synthesis of high molecular weight RNA. Processing of ribosomal precursor RNA was altered. these results suggest that the primary effect of OP and HQ is on rRNA synthesis. RNA metabolism must therefore have a key role in the regulation of the cell cycle.     

Metastasis suppression by adiponectin: LKB1 rises up to the challenge

Adiponectin is an adipocytokine involved in the pathogenesis of various obesity-related disorders. Also, it has been shown that adiponectin has therapeutic potential for metabolic syndrome, systemic insulin resistance, cardiovascular disease and more recently carcinogenesis. Adiponectin can modulate breast cancer cell growth and proliferation. Anti-metastatic effects of adiponectin have also been elucidated. It has been shown that adiponectin inhibits important metastatic properties such as adhesion, invasion and migration of breast cancer cells. Examination of the underlying molecular mechanisms has shown that adiponectin treatment increases AMP-activated protein kinase (AMPK) phosphorylation and activity. Adiponectin also increases phosphorylation of downstream target of AMPK, Acetyl-CoA Carboxylase (ACC) and decreases phosphorylation of p70S6 kinase (S6K). Importantly, adiponectin treatment increases the expression of tumor suppressor gene, LKB1 in breast cancer cells. LKB1 is required for adiponectin-mediated modulation of AMPK-S6K axis and more importantly, its biological functions including inhibition of adhesion, migration and invasion of breast cancer cells. Although further studies are required to analyze the effect of adiponectin on LKB1-AMPK-S6K axis, these data present a novel mechanism involving specific upregulation of tumor suppressor gene LKB1 by which adiponectin inhibits adhesion, invasion and migration of breast cancer cells. These results highlight a new role for LKB1 in adiponectin action and may have significant implication for development of novel therapeutic options.Cancer research has largely focused on the molecular basis of oncogenic transformation and tumorigenesis for many years. Recent progress in cancer research has put the metastatic process at the center stage because higher metastatic potential of tumor cells is the major cause of mortality from solid tumors. Metastasis is a complex process that involves modulation of various molecular signaling networks. Tumor cells alter the microenvironment, attain greater cellular adhesion along with better ability to invade and migrate to gain access to circulation. These wandering tumor cells defy anoikis, survive in the circulation, exit into new permissive organ site and colonize distant organs.¹ The microenvironment in which the tumor originates plays an important role in tumor initiation, progression and metastasis.Key words: adiponectin, LKB1, invasion, migration, cancer, AMPK, S6K     

Down regulation of the alpha-factor pheromone receptor in S. cerevisiae

The peptide pheromone, alpha-factor, was found to elicit down regulation of receptor sites on yeast a cell targets. Cellular uptake of alpha-factor accompanied the loss of receptor sites. Receptor-deficient a cells bearing a deletion of the STE2 gene were unable to internalize alpha-factor. Cultures were found to reaccumulate receptor sites following the initial period of down regulation; reaccumulation was dependent upon protein synthesis. Pheromone-resistant mutants, ste4-3 and ste5-3, retained the ability to down regulate receptors but failed to show reaccumulation. Our results suggest that alpha-factor-receptor complexes enter the cell by receptor-mediated endocytosis and that receptors are continuously lost and resynthesized in the presence of alpha-factor. We found no reduction of alpha-factor binding capacity in a cell cultures that had adapted to alpha-factor.     

Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS.

Diploid S. cerevisiae strains undergo a dimorphic transition that involves changes in cell shape and the pattern of cell division and results in invasive filamentous growth in response to starvation for nitrogen. Cells become long and thin and form pseudohyphae that grow away from the colony and invade the agar medium. Pseudohyphal growth allows yeast cells to forage for nutrients. Pseudohyphal growth requires the polar budding pattern of a/alpha diploid cells; haploid axially budding cells of identical genotype cannot undergo this dimorphic transition. Constitutive activation of RAS2 or mutation of SHR3, a gene required for amino acid uptake, enhance the pseudohyphal phenotype; a dominant mutation in RSR1/BUD1 that causes random budding suppresses pseudohyphal growth.     

Targeting of the mammalian nucleoporin p62 to the nuclear envelope in the yeast Saccharomyces cerevisiae and HeLa cells.

We have analyzed the sorting of the mammalian nucleoporin p62 in human culture cells and in the yeast Saccharomyces cerevisiae. To this end, gene fusions were generated that carry Aequorea victoria green fluorescence protein and defined portions of p62. Upon transient gene expression fluorescent fusion proteins were localized in HeLa cells. Likewise, fusion proteins were studied in S. cerevisiae using wild-type as well as mutant cells that cluster nuclear pore complexes. Our results demonstrate that evolutionarily distant organisms, such as humans and yeasts, recognize the same sequence elements of p62 for sorting to the nuclear envelope. Specifically, the entire sequence of p62 or its complete C-terminal domain targeted fusion proteins to the nuclear membranes. In contrast, truncations of the C-terminal domain or the N-terminal segment of p62 failed to associate with the nuclear envelope in either organism. In HeLa cells overexpression of several p62-containing fusion proteins resulted in nuclear fragmentation. The C-terminal domain of p62 caused this effect, and amino acid residues 477 to 525 were sufficient to induce aberrant nuclei. Thus, overexpression of 49 amino acid residues located at the C-terminal tail of p62 interferes with the nuclear integrity in human culture cells.     

Functional expression of mammalian cytochromes P450IIB in the yeast Saccharomyces cerevisiae

Three mammalian cytochromes P450 from the IIB subfamily, P450IIB11 from canine and P450IIB4 and P450IIB5 from rabbit, have been expressed in the yeast Saccharomyces cerevisiae by use of an autonomously replicating vector containing the galactose-inducible gal10 promoter. Cytochromes P450IIB4 and P450IIB5 are closely related proteins, with only 11 amino acid substitutions between them. P450IIB11 is a homologous protein, likely orthologous with IIB4 or IIB5, with 102 amino acid substitutions compared with the P450IIB4 protein and 106 compared with the P450IIB5 protein. The expressed proteins are functional in yeast microsomes, exhibiting activity toward androstenedione, 7-ethoxycoumarin, and, in some cases, progesterone. Expressed cytochromes P450IIB4 and P450IIB11 hydroxylate androstenedione with regio- and stereoselectivity characteristic of the purified, reconstituted proteins. A striking difference in the androstenedione metabolite profiles of IIB4 and IIB5 was observed, with IIB4 producing almost exclusively the 16 beta-hydroxy metabolite and IIB5 producing the 16 alpha-hydroxy and 15 alpha-hydroxy products. This is the first time that 15 alpha-hydroxylase activity has been associated with IIB4/IIB5. This activity has also been detected in liver microsomes from some, but not all, individual phenobarbital-induced rabbits tested and is largely inhibited by anti-rabbit P450IIB immunoglobulin G. These studies illustrate the utility of the yeast expression system for defining catalytic activities of individual mammalian cytochromes P450 and identifying new marker activities that can be utilized in liver microsomes.     

The role of hexose transport and phosphorylation in cAMP signalling in the yeast Saccharomyces cerevisiae

Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation. The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose. Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling. Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis. Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling. This indicates that a regulatory function may be associated with the hexose kinases. Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase. This may indicate that intracellular acidification can boost a downstream effect that amplifies the residual signal transmitted via the hexose kinases when glucose uptake is too low.     

Tandemly arranged variant 5S ribosomal RNA genes in the yeast Saccharomyces cerevisiae.

Most of the ribosomal RNA genes of the yeast Saccharomyces cerevisiae are about 9 kilobases (kb) in size and encode both the 35S rRNA (processed to produce the 25S, 18S, and 5.8S species) and 5S rRNA. These genes are arranged in a single tandem array of 100 repeats. Below, we present evidence that at the centromere-distal end of this array is a tandem arrangement of a different type of rRNA gene. Each of these repeats is 3.6 kb in length and encodes a single 5S rRNA. The coding sequence of this gene is different from that of the "normal" 5S gene in three positions located at the 3' end of the gene.     

Transfer of yeast artificial chromosomes from yeast to mammalian cells.

Human DNA can be cloned as yeast artificial chromosomes (YACs), each of which contains several hundred kilobases of human DNA. This DNA can be manipulated in the yeast host using homologous recombination and yeast selectable markers. In relatively few steps it is possible to make virtually any change in the cloned human DNA from single base pair changes to deletions and insertions. In order to study the function of the cloned DNA and the effects of the changes made in the yeast, the human DNA must be transferred back into mammalian cells. Recent experiments indicate that large genes can be transferred from the yeast host to mammalian cells in tissue culture and that the genes are transferred intact and are expressed. Using the same methods it may soon be possible to transfer YAC DNA into the mouse germ line so that the expression and function of genes cloned in YACs can be studied in developing and adult mammalian animals.