Protracted volitional spawning of pinfish Lagodon rhomboides and changes in egg quality and fatty-acid composition throughout the spawning season.

Spawning performance of pinfish Lagodon rhomboides without use of hormonal aids was monitored over an extended season. Nearly three million eggs were obtained from 75 spawns collected over a 90-day consecutive period from a single population of four brood fish (1M:1F). A mean ± s.d. batch fecundity of 30·27 ± 22·64 eggs g-1 female was estimated with 98·0 ± 0·06% of the batch composed of floating eggs which were 1·04 ± 0·04 mm in diameter and 85·71 ± 27·59% fertile. Floating eggs successfully hatched 54·65 ± 29·13% of the time which yielded larvae that were 2·59 ± 0·24 mm in length. Fatty acids within floating eggs were largely represented by polyunsaturated fatty acids (45·30 ± 2·14% of total fatty acids) of which linoleic acid [(c18:2n-6cis) 3·49 ± 1·69% trifluoroacetic acid (TFA)] and docosahexaenoic acid (DHA) [(c22:6n-3) 28·47 ± 1·48% TFA] represented the majority of fatty acids for n-6 and n-3 polyunsaturated fatty acids, respectively. The strongest correlations between fatty acids and hatching success and larval survival to first feeding were observed for the DHA:EPA (eicosapentaenoic acid; c20:5n-3) ratio and total n-6 polyunsaturated fatty-acids levels, respectively. These data demonstrate potential for producers to rely on natural spawns for extensive egg production and provide a baseline for future development of natural spawning protocols of captive L. rhomboides.

Do cells think?

A microorganism has to adapt to changing environmental conditions in order to survive. Cells could follow one of two basic strategies to address such environmental fluctuations. On the one hand, cells could anticipate a fluctuating environment by spontaneously generating a phenotypically diverse population of cells, with each subpopulation exhibiting different capacities to flourish in the different conditions. Alternatively, cells could sense changes in the surrounding conditions - such as temperature, nutritional availability or the presence of other individuals - and modify their behavior to provide an appropriate response to that information. As we describe, examples of both strategies abound among different microorganisms. Moreover, successful application of either strategy requires a level of memory and information processing that has not been normally associated with single cells, suggesting that such organisms do in fact have the capacity to 'think'.

The role of phosphatases in TOR signaling in yeast.

The TOR pathway controls cellular functions necessary for cell growth and proliferation of yeast and larger eukaryotes. The search for members of the TOR signaling cascade in yeast led to the discovery of type 2A protein phosphatases (PP2A) as important players within the pathway. We describe the roles in yeast of PP2A and the closely related phosphatase, Sit4, and then focus on complexes formed between the catalytic subunit of these phosphatases and Tap42, a direct target of the Tor protein kinases in yeast. Recent results suggest that Tap42 mediates many of the Tor functions in yeast, especially those involved in transcriptional modulation. However, whether Tap42 executes its function by inhibiting phosphatase activity or by activating phosphatases is still uncertain. In addition, Tor affects some transcriptional and physiological processes through Tap42 independent pathways. Thus, Tor proteins use multiple mechanisms to regulate transcriptional and physiological processes in yeast.

Genetic selection in Saccharomyces of mutant mammalian adenylyl cyclases with elevated basal activities.

We show that co-expression of rat Galphas together with type I, II, IV, or VI mammalian adenylyl cyclase (AC) can suppress the growth defect of cyr1 strains of Saccharomyces cerevisiae, which lack a functional endogenous AC. Complemention of cvr1 is not observed in the absence of Galphas, indicating that the mammalian ACs retain their normal regulatory behavior in yeast. Selection for Galphas-independent growth of (cyr1 strains expressing type IV AC yielded several ACIV mutants with enhanced basal activity, each of which had a single amino acid substitution in the conserved C1a or C2a region of the protein. Expression of two of the mutant ACs in HEK293 cells resulted in increased levels of cAMP and elevated adenylyl cyclase activity. Further selection for reverting mutations in one of these constitutively active AC mutants yielded three independent intragenic suppressor mutations. The distribution of the activating and suppressor mutations throughout both C1a and C2a is consistent with a model in which the enhanced basal activity results from an increase in the affinity between C1a and C2a. These results demonstrate the utility of Saccharomyces as a tool for the identification of informative mutant forms of mammalian ACs.

Sensitivity of wild type and mutant ras alleles to Ras specific exchange factors: Identification of factor specific requirements.

We have investigated the productive interaction between the four mammalian Ras proteins (H-, N-, KA- and KB-Ras) and their activators, the mammalian exchange factors mSos1, GRF1 and GRP, by using a modified Saccharomyces cerevisiae whose growth is dependent on activation of a mammalian Ras protein by its activator. All four mammalian Ras proteins were activated with similar efficiencies by the individual exchange factors. The H-Ras mutant V103E, which is competent for membrane localization, nucleotide binding, intrinsic and stimulated GTPase activity as well as intrinsic exchange, was defective for activation by all factors tested, suggesting that the integrity of this residue is necessary for catalyzed exchange. However, when other H-Ras mutants were studied, some distinct sensitivities to the exchange factors were observed. GRP-mediated, but not mSos1-mediated, exchange was blocked in additional mutants, suggesting different structural requirements for GRP. Analysis of Ras-mediated gene activation in murine fibroblasts confirmed these results.

Chromosomal boundaries in S. cerevisiae.

Chromatin boundary elements or insulators in metazoans delimit distinct chromosomal domains of gene expression. Recently, DNA sequences with properties similar to boundary elements were also discovered in Saccharomyces cerevisiae. These sequences block the spread of transcriptionally silent chromatin, the yeast equivalent of metazoan heterochromatin, and are referred to as 'heterochromatin barriers'. These barriers share no sequence homology but all consist of multiple binding sites for various regulatory proteins. Current data suggest that barriers may function in yeast by recruiting a protein complex that precludes nucleosome assembly and thereby disrupts a contiguous array of nucleosomes required for the spread of silent chromatin.

CAC3(MSI1) suppression of RAS2(G19V) is independent of chromatin assembly factor I and mediated by NPR1.

Cac3p/Msi1p, the Saccharomyces cerevisiae homolog of retinoblastoma-associated protein 48 (RbAp48), is a component of chromatin assembly factor I (CAF-I), a complex that assembles histones H3 and H4 onto replicated DNA. CAC3 overexpression also suppresses the RAS/cyclic AMP (cAMP) signal transduction pathway by an unknown mechanism. We investigated this mechanism and found that CAC3 suppression of RAS/cAMP signal transduction was independent of either CAC1 or CAC2, subunits required for CAF-I function. CAC3 suppression was also independent of other chromatin-modifying activities, indicating that Cac3p has at least two distinct, separable functions, one in chromatin assembly and one in regulating RAS function. Unlike Cac1p, which localizes primarily to the nucleus, Cac3p localizes to both the nucleus and the cytoplasm. In addition, Cac3p associates with Npr1p, a cytoplasmic kinase that stablizes several nutrient transporters by antagonizing a ubiquitin-mediated protein degradation pathway. Deletion of NPR1, like overexpression of Cac3p, suppressed the RAS/cAMP pathway. Furthermore, NPR1 overexpression interfered with the ability of CAC3 to suppress the RAS/cAMP pathway, indicating that extra Cac3p suppresses the RAS/cAMP pathway by sequestering Npr1p. Deletion of NPR1 did not affect the quantity, phosphorylation state, or localization of Ras2p. Consistent with the idea that Npr1p exerts its effect on the RAS/cAMP pathway by antagonizing a ubiquitin-mediated process, excess ubiquitin suppressed both the heat shock sensitivity and the sporulation defects caused by constitutive activation of the RAS/cAMP pathway. Thus, CAC3/MSI1 regulates the RAS/cAMP pathway via a chromatin-independent mechanism that involves the sequestration of Npr1p and may be due to the increased ubiquitination of an Npr1p substrate.

Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo.

The phosphoprotein phosphatase 2A (PP2A) catalytic subunit contains a methyl ester on its C-terminus, which in mammalian cells is added by a specific carboxyl methyltransferase and removed by a specific carboxyl methylesterase. We have identified genes in yeast that show significant homology to human carboxyl methyltransferase and methylesterase. Extracts of wild-type yeast cells contain carboxyl methyltransferase activity, while extracts of strains deleted for one of the methyltransferase genes, PPM1, lack all activity. Mutation of PPM1 partially disrupts the PP2A holoenzyme in vivo and ppm1 mutations exhibit synthetic lethality with mutations in genes encoding the B or B' regulatory subunit. Inactivation of PPM1 or overexpression of PPE1, the yeast gene homologous to bovine methylesterase, yields phenotypes similar to those observed after inactivation of either regulatory subunit. These phenotypes can be reversed by overexpression of the B regulatory subunit. These results demonstrate that Ppm1 is the sole PP2A methyltransferase in yeast and that its activity is required for the integrity of the PP2A holoenzyme.

The yeast HML I silencer defines a heterochromatin domain boundary by directional establishment of silencing.

The eukaryotic genome is divided into functional domains defined in part by local differences in chromatin structure and delimited in many cases by boundary elements. The HML and HMR loci in the yeast Saccharomyces cerevisiae are transcriptionally silent chromosome domains. Each locus is bracketed by two cis-acting sequences, designated E and I, that serve to establish and maintain repression of genes within each locus. We show that repression at HML is uniformly high between E and I but decreases sharply beyond I. The region of repression at HML generally correlates with the domain of histone hypoacetylation. Despite the sharp definition of the boundaries of HML, no sequence capable of blocking the spread of heterochromatin resides in the sequences flanking HML. We find, though, that inverting the orientation of I increases silencing outside of HML while weakening silencing within HML. These results indicate that the HML I silencer establishes a boundary between active and inactive chromatin at HML, but does so by organizing inactive chromatin in only one direction. This represents a different mechanism for delimiting the boundaries of a eukaryotic chromosome domain.

UASrpg can function as a heterochromatin boundary element in yeast.

The HM loci in Saccharomyces cerevisiae constitute region-specific but gene-nonspecific repression domains, as a number of heterologous genes transcribed by RNA polymerase II or III are silenced when placed at these loci. The promoters of the Ashbya gossypii TEF gene and the S. cerevisiae TEF1 and TEF2 genes, however, are resistant to transcriptional silencing by the HM silencers in yeast. Moreover, when interposed between the HML alpha genes and the E silencer, certain segments of these promoters block the repression effect of the silencer on the alpha genes. All of these fragments contain UASrpg (upstream activation sequence of ribosome protein genes) composed of multiple binding sites for Rap1. In fact, a 149-bp segment consisting essentially of only three tandem Rap1-binding sites from the UASrpg of yeast TEF2 exhibits silencer-blocking activity. This element also exhibits insulating activity and orientation dependence characteristic of known chromatin boundary elements. Finally, the element blocks the physical spread of heterochromatin initiated at a silencer. This segment provides the first example of chromatin domain boundary or insulator elements in yeast.

Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast.

Tor proteins, homologous to DNA-dependent protein kinases, participate in a signal transduction pathway in yeast that regulates protein synthesis and cell wall expansion in response to nutrient availability. The anti-inflammatory drug rapamycin inhibits yeast cell growth by inhibiting Tor protein signaling. This leads to diminished association of a protein, Tap42, with two different protein phosphatase catalytic subunits; one encoded redundantly by PPH21 and PPH22, and one encoded by SIT4. We show that inactivation of either Cdc55 or Tpd3, which regulate Pph21/22 activity, results in rapamycin resistance and that this resistance correlates with an increased association of Tap42 with Pph21/22. Furthermore, we show Tor-dependent phosphorylation of Tap42 both in vivo and in vitro and that this phosphorylation is rapamycin sensitive. Inactivation of Cdc55 or Tpd3 enhances in vivo phosphorylation of Tap42. We conclude that Tor phosphorylates Tap42 and that phosphorylated Tap42 effectively competes with Cdc55/Tpd3 for binding to the phosphatase 2A catalytic subunit. Furthermore, Cdc55 and Tpd3 promote dephosphorylation of Tap42. Thus, Tor stimulates growth-promoting association of Tap42 with Pph21/22 and Sit4, while Cdc55 and Tpd3 inhibit this association both by direct competition and by dephosphorylation of Tap42. These results establish Tap42 as a target of Tor and add further refinement to the Tor signaling pathway.

Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeast.

We describe a procedure for isolating agonists for mammalian G protein-coupled receptors of unknown function. Human formyl peptide receptor like-1 (FPRL-1) receptor, originally identified as an orphan G protein-coupled receptor related to the formyl peptide receptor (FPR1), was expressed in Saccharomyces cells designed to couple receptor activation to histidine prototrophy. Selection for histidine prototrophs among transformants obtained with a plasmid-based library encoding random peptides identified six different agonists, each of whose production yielded autocrine stimulation of the receptor expressed in yeast. A synthetic version of each peptide promoted activation of FPRL-1 expressed in human embryonic kidney (HEK293) cells, and five of the peptides exhibited significant selectivity for activation of FPRL-1 relative to FPR1. One selective peptide was tested and found to mobilize calcium in isolated human neutrophils. This demonstrates that stimulation of FPRL-1 results in neutrophil activation and suggests that the receptor functions as a component of the inflammatory response. This autocrine selection protocol may be a generally applicable method for providing pharmacological tools to evaluate the physiological roles of the growing number of mammalian orphan G protein-coupled receptors.

Efficient transition to growth on fermentable carbon sources in Saccharomyces cerevisiae requires signaling through the Ras pathway.

Strains carrying ras2(318S) as their sole RAS gene fail to elicit a transient increase in cAMP levels following addition of glucose to starved cells but maintain normal steady-state levels of cAMP under a variety of growth conditions. Such strains show extended delays in resuming growth following transition from a quiescent state to glucose-containing growth media, either in emerging from stationary phase or following inoculation as spores onto fresh media. Otherwise, growth of such strains is indistinguishable from that of RAS2(+) strains. ras2(318S) strains also exhibit a delay in glucose-stimulated phosphorylation and turnover of fructose-1,6-bisphosphatase, a substrate of the cAMP-dependent protein kinase A (PKA) and a key component of the gluconeogenic branch of the glycolytic pathway. Finally Tpk(w) strains, which fail to modulate PKA in response to fluctuations in cAMP levels, show the same growth delay phenotypes, as do ras2(318S) strains. These observations indicate that the glucose-induced cAMP spike results in a transient activation of PKA, which is required for efficient transition of yeast cells from a quiescent state to resumption of rapid growth. This represents the first demonstration that yeast cells use the Ras pathway to transmit a signal to effect a biological change in response to an upstream stimulus.

New assay technologies for high-throughput screening.

The use of high-throughput screening for early stage drug discovery imposes several constraints on the format of assays for therapeutic targets of interest. Homogeneous cell-free assays based on energy transfer, fluorescence polarization spectroscopy or fluorescence correlation spectroscopy provide the sensitivity, ease, speed and resistance to interference from test compounds needed to function in a high-throughput screening mode. Similarly, novel cell-based assays are now being adapted for high-throughput screening, providing for in situ analysis of a variety of biological targets. Finally, recent advances in assay miniaturization mark a transition to ultra high-throughput screening, ensuring that identification of lead compounds will not be the rate-limiting step in finding new drugs.

p53 mutations isolated in yeast based on loss of transcription factor activity: similarities and differences from p53 mutations detected in human tumors.

p53 is a transcriptional activator that plays a key role in the integration of signals inducing cell division arrest and programmed cell death. Moreover, p53 is a tumor suppressor gene, mutations of which are the most commonly detected mutations in diverse malignancies. In order to better understand the significance of p53 mutations to human cancer, we isolated mutant alleles of p53 that had lost transcription factor activity in yeast. These mutant alleles were evaluated for their precise changes, their activity against three different p53 responsive enhancers and their ability to act in a transdominant fashion to the wild type allele. While many of the mutations isolated in yeast resembled those found in human tumors, consistent with the importance of transcription factor activity for p53 in mammalian cells, the mutational spectrum obtained was dependent upon the p53 enhancer employed for the selection. Some mutations specifically inactivated p53 in yeast for a single enhancer element. Virtually all missense mutations tested had a dominant inhibitory effect on wild type p53 in yeast. Since some of these transdominant mutations are virtually unknown in human tumors we conclude that transdominance, per se, fails to predict which mutations occur frequently in cancer.

Sphingoid base 1-phosphate phosphatase: a key regulator of sphingolipid metabolism and stress response.

The sphingolipid metabolites ceramide and sphingosine-1-phosphate are second messengers with opposing roles in mammalian cell growth arrest and survival; their relative cellular level has been proposed to be a rheostat that determines the fate of cells. This report demonstrates that this rheostat is an evolutionarily conserved stress-regulatory mechanism that influences growth and survival of yeast. Although the role of sphingosine-1-phosphate in yeast was not previously examined, accumulation of ceramide has been shown to induce G1 arrest and cell death. We now have identified a gene in Saccharomyces cerevisiae, LBP1, that regulates the levels of phosphorylated sphingoid bases and ceramide. LBP1 was cloned from a yeast mutant that accumulated phosphorylated long-chain sphingoid bases and diverted sphingoid base intermediates from sphingolipid pathways to glycerophospholipid biosynthesis. LBP1 and its homolog, LBP2, encode very hydrophobic proteins that contain a novel-conserved sequence motif for lipid phosphatases, and both have long-chain sphingoid base phosphate phosphatase activity. In vitro characterization of Lbp1p shows that this phosphatase is Mg2+-independent with high specificity for phosphorylated long-chain bases, phytosphingosine and sphingosine. The deletion of LBP1 results in the accumulation of phosphorylated long-chain sphingoid bases and reduced ceramide levels. Moreover, deletion of LBP1 and LBP2 results in dramatically enhanced survival upon severe heat shock. Thus, these phosphatases play a previously unappreciated role in regulating ceramide and phosphorylated sphingoid base levels in yeast, and they modulate stress responses through sphingolipid metabolites in a manner that is reminiscent of their effects on mammalian cells.

DNA in transcriptionally silent chromatin assumes a distinct topology that is sensitive to cell cycle progression.

Transcriptionally silent regions of the Saccharomyces cerevisiae genome, the silent mating type loci and telomeres, represent the yeast equivalent of metazoan heterochromatin. To gain insight into the nature of silenced chromatin structure, we have examined the topology of DNA spanning the HML silent mating type locus by determining the superhelical density of mini-circles excised from HML (HML circles) by site-specific recombination. We observed that HML circles excised in a wild-type (SIR+) strain were more negatively supercoiled upon deproteinization than were the same circles excised in a sir- strain, in which silencing was abolished, even when HML alleles in which neither circle was transcriptionally competent were used. cis-acting sites flanking HML, called silencers, are required in the chromosome for establishment and inheritance of silencing. HML circles excised without silencers from cells arrested at any point in the cell cycle retained SIR-dependent differences in superhelical density. However, progression through the cell cycle converted SIR+ HML circles to a form resembling that of circles from sir- cells. This decay was not observed with circles carrying a silencer. These results establish that (i) DNA in transcriptionally silenced chromatin assumes a distinct topology reflecting a distinct organization of silenced versus active chromatin; (ii) the altered chromatin structure in silenced regions likely results from changes in packaging of individual nucleosomes, rather than changes in nucleosome density; and (iii) cell cycle progression disrupts the silenced chromatin structure, a process that is counteracted by silencers.

Alpha2p controls donor preference during mating type interconversion in yeast by inactivating a recombinational enhancer of chromosome III.

Homothallic strains of Saccharomyces cerevisiae can change mating type as often as every generation by replacing the allele at the MAT locus with a copy of mating type information present at one of two storage loci, HML and HMR, located on either end of chromosome III. Selection of the appropriate donor locus is dictated by a mating type-specific repressor protein, alpha2p: Cells containing alpha2p select HMR, whereas those lacking alpha2p select HML. As a repressor protein, alpha2p binds to DNA cooperatively with the transcriptional activator Mcm1p. Here we show that two alpha2p/Mcm1p-binding sites, DPS1 and DPS2, control donor selection. DPS1 and DPS2 are located approximately 30 kb from the left arm of chromosome III, well removed from HML, HMR, and MAT. Precise deletion of only DPS1 and DPS2 results in random selection of donor loci and in a cells without affecting selection in alpha cells. Reciprocally, deletion of only the alpha2p binding segments in each of these two sites results in selection of the wrong donor loci in alpha cells without affecting preference in a cells. These results suggest that Mcm1p, bound to these two sites in the absence of alpha2p, activates HML as donor. Binding of alpha2p blocks the ability of Mcm1p bound to DPS1 and DPS2 to activate HML, resulting in default selection of HMR as donor. DPS1 and DPS2 also regulate expression of several noncoding RNAs, although deletion of at least one of these RNA loci does not affect donor preference. This suggests that transcriptional activation, rather than transcription of a specific product, is the initiating event in activating the left arm of chromosome III for donor selection.