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Meiosis-specific chromatin structures, guided by histone modifications, are critical mediators of a meiotic transient transcription program and progression through prophase I. Histone H3K4 can be methylated up to three times by the Set1-containing COMPASS complex and each methylation mark corresponds to a different chromatin conformation. The level of H3K4 modification is directed by the activity of additional COMPASS components. In this study, we characterized the role of the COMPASS subunits during meiosis in Saccharomyces cerevisiae. In vegetative cells, previous studies revealed a role for subunits Swd2, Sdc1, and Bre2 for H3K4me2 while Spp1 supported trimethylation. However, we found that Bre2 and Sdc1 are required for H3K4me3 as yeast prepare to enter meiosis while Spp1 is not. Interestingly, we identified distinct meiotic functions for the core COMPASS complex members that required for all H3K4me, Set1, Swd1, and Swd3. While Set1 and Swd1 are required for progression through early meiosis, Swd3 is critical for late meiosis and spore morphogenesis. Furthermore, the meiotic requirement for Set1 is independent of H3K4 methylation, suggesting the presence of nonhistone substrates. Finally, checkpoint suppression analyses indicate that Set1 and Swd1 are required for both homologous recombination and chromosome segregation. These data suggest that COMPASS has important new roles for meiosis that are independent of its well-characterized functions during mitotic divisions., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

In the budding yeast Saccharomyces cerevisiae , nutrient depletion induces massive transcriptional reprogramming that relies upon communication between transcription factors, post-translational histone modifications, and the RNA polymerase II holoenzyme complex. Histone H3Lys4 methylation (H3Lys4 me), regulated by the Set1p-containing COMPASS methyltransferase complex and Jhd2p demethylase, is one of the most well-studied histone modifications. We previously demonstrated that the RNA polymerase II mediator components cyclin C-Cdk8p inhibit locus-specific H3Lys4 3me independently of Jhd2p Here, we identify loci subject to cyclin C- and Jhd2p-dependent histone H3Lys4 3me inhibition using chromatin immunoprecipitation (ChIP)-seq. We further characterized the independent and combined roles of cyclin C and Jhd2p in controlling H3Lys4 3me and transcription in response to fermentable and nonfermentable carbon at multiple loci. These experiments suggest that H3Lys4 3me alone is insufficient to induce transcription. Interestingly, we identified an unexpected role for cyclin C-Cdk8p in repressing AQY1 transcription, an aquaporin whose expression is normally induced during nutrient deprivation. These experiments, combined with previous work in other labs, support a two-step model in which cyclin C-Cdk8p mediate AQY1 transcriptional repression by stimulating transcription factor proteolysis and preventing Set1p recruitment to the AQY1 locus., (Copyright © 2017 Law and Finger.)

Chromatin modification enzymes are important regulators of gene expression and some are evolutionarily conserved from yeast to human. Saccharomyces cerevisiae is a major model organism for genome-wide studies that aim at the identification of target genes under the control of conserved epigenetic regulators. Ume6 interacts with the upstream repressor site 1 (URS1) and represses transcription by recruiting both the conserved histone deacetylase Rpd3 (through the co-repressor Sin3) and the chromatin-remodeling factor Isw2. Cells lacking Ume6 are defective in growth, stress response, and meiotic development. RNA profiling studies and in vivo protein-DNA binding assays identified mRNAs or transcript isoforms that are directly repressed by Ume6 in mitosis. However, a comprehensive understanding of the transcriptional alterations, which underlie the complex ume6Δ mutant phenotype during fermentation, respiration, or sporulation, is lacking. We report the protein-coding transcriptome of a diploid MAT a/α wild-type and ume6/ume6 mutant strains cultured in rich media with glucose or acetate as a carbon source, or sporulation-inducing medium. We distinguished direct from indirect effects on mRNA levels by combining GeneChip data with URS1 motif predictions and published high-throughput in vivo Ume6-DNA binding data. To gain insight into the molecular interactions between successive waves of Ume6-dependent meiotic genes, we integrated expression data with information on protein networks. Our work identifies novel Ume6 repressed genes during growth and development and reveals a strong effect of the carbon source on the derepression pattern of transcripts in growing and developmentally arrested ume6/ume6 mutant cells. Since yeast is a useful model organism for chromatin-mediated effects on gene expression, our results provide a rich source for further genetic and molecular biological work on the regulation of cell growth and cell differentiation in eukaryotes.

BOI1 and BOI2 are paralogs important for the actin cytoskeleton and polar growth. BOI1 encodes a meiotic transcript isoform with an extended 5'-untranslated region predicted to impair protein translation. It is, however, unknown how the isoform is repressed during mitosis, and if Boi1 is present during sporulation. By interpreting microarray data from MATa cells, MATa/α cells, a starving MATα/α control, and a meiosis-impaired rrp6 mutant, we classified BOI1's extended isoform as early meiosis-specific. These results were confirmed by RNA-Sequencing, and extended by a 5'-RACE assay and Northern blotting, showing that meiotic cells induce the long isoform while the mitotic isoform remains detectable during meiosis. We provide evidence via motif predictions, an in vivo binding assay and genetic experiments that the Rpd3/Sin3/Ume6 histone deacetylase complex, which represses meiotic genes during mitosis, also prevents the induction of BOI1's 5'-extended isoform in mitosis by direct binding of Ume6 to its URS1 target. Finally, we find that Boi1 protein levels decline when cells switch from fermentation to respiration and sporulation. The histone deacetylase Rpd3 is conserved, and eukaryotic genes frequently encode transcripts with variable 5'-UTRs. Our findings are therefore relevant for regulatory mechanisms involved in the control of transcript isoforms in multi-cellular organisms., (© 2015 John Wiley & Sons Ltd.)

Diploid budding yeast undergoes rapid mitosis when it ferments glucose, and in the presence of a non-fermentable carbon source and the absence of a nitrogen source it triggers sporulation. Rich medium with acetate is a commonly used pre-sporulation medium, but our understanding of the molecular events underlying the acetate-driven transition from mitosis to meiosis is still incomplete. We identified 263 proteins for which mRNA and protein synthesis are linked or uncoupled in fermenting and respiring cells. Using motif predictions, interaction data and RNA profiling we find among them 28 likely targets for Ume6, a subunit of the conserved Rpd3/Sin3 histone deacetylase-complex regulating genes involved in metabolism, stress response and meiosis. Finally, we identify 14 genes for which both RNA and proteins are detected exclusively in respiring cells but not in fermenting cells in our sample set, including CSM4, SPR1, SPS4 and RIM4, which were thought to be meiosis-specific. Our work reveals intertwined transcriptional and post-transcriptional control mechanisms acting when a MATa/α strain responds to nutritional signals, and provides molecular clues how the carbon source primes yeast cells for entering meiosis., Biological Significance: Our integrated genomics study provides insight into the interplay between the transcriptome and the proteome in diploid yeast cells undergoing vegetative growth in the presence of glucose (fermentation) or acetate (respiration). Furthermore, it reveals novel target genes involved in these processes for Ume6, the DNA binding subunit of the conserved histone deacetylase Rpd3 and the co-repressor Sin3. We have combined data from an RNA profiling experiment using tiling arrays that cover the entire yeast genome, and a large-scale protein detection analysis based on mass spectrometry in diploid MATa/α cells. This distinguishes our study from most others in the field-which investigate haploid yeast strains-because only diploid cells can undergo meiotic development in the simultaneous absence of a non-fermentable carbon source and nitrogen. Indeed, we report molecular clues how respiration of acetate might prime diploid cells for efficient spore formation, a phenomenon that is well known but poorly understood., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The tripartite Rpd3/Sin3/Ume6 complex represses meiotic isoforms during mitosis. We asked if it also controls starvation-induced isoforms. We report that VTH1/VTH2 encode acetate-inducible isoforms with extended 5'-regions overlapping antisense long non-coding RNAs. Rpd3 and Ume6 repress the long isoform of VTH2 during fermentation. Cells metabolising glucose contain Vth2, while the protein is undetectable in acetate and during sporulation. VTH2 is a useful model locus to study mechanisms implicating promoter directionality, lncRNA transcription and post-transcriptional control of gene expression via 5'-UTRs. Since mammalian genes encode transcript isoforms and Rpd3 is conserved, our findings are relevant for gene expression in higher eukaryotes., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Transcriptional regulation is dependent upon the interactions between the RNA pol II holoenzyme complex and chromatin. RNA pol II is part of a highly conserved multiprotein complex that includes the core mediator and CDK8 subcomplex. In Saccharomyces cerevisiae, the CDK8 subcomplex, composed of Ssn2p, Ssn3p, Ssn8p, and Srb8p, is thought to play important roles in mediating transcriptional control of stress-responsive genes. Also central to transcriptional control are histone post-translational modifications. Lysine methylation, dynamically balanced by lysine methyltransferases and demethylases, has been intensively studied, uncovering significant functions in transcriptional control. A key question remains in understanding how these enzymes are targeted during stress response. To determine the relationship between lysine methylation, the CDK8 complex, and transcriptional control, we performed phenotype analyses of yeast lacking known lysine methyltransferases or demethylases in isolation or in tandem with SSN8 deletions. We show that the RNA pol II CDK8 submodule components SSN8/SSN3 and the histone demethylase JHD2 are required to inhibit pseudohyphal growth-a differentiation pathway induced during nutrient limitation-under rich conditions. Yeast lacking both SSN8 and JHD2 constitutively express FLO11, a major regulator of pseudohyphal growth. Interestingly, deleting known FLO11 activators including FLO8, MSS11, MFG1, TEC1, SNF1, KSS1, and GCN4 results in a range of phenotypic suppression. Using chromatin immunoprecipitation, we found that SSN8 inhibits H3 Lys4 trimethylation independently of JHD2 at the FLO11 locus, suggesting that H3 Lys4 hypermethylation is locking FLO11 into a transcriptionally active state. These studies implicate the CDK8 subcomplex in fine-tuning H3 Lys4 methylation levels during pseudohyphal differentiation., (Copyright © 2015 by the Genetics Society of America.)

It was recently reported that the sizes of many mRNAs change when budding yeast cells exit mitosis and enter the meiotic differentiation pathway. These differences were attributed to length variations of their untranslated regions. The function of UTRs in protein translation is well established. However, the mechanism controlling the expression of distinct transcript isoforms during mitotic growth and meiotic development is unknown. In this study, we order developmentally regulated transcript isoforms according to their expression at specific stages during meiosis and gametogenesis, as compared to vegetative growth and starvation. We employ regulatory motif prediction, in vivo protein-DNA binding assays, genetic analyses and monitoring of epigenetic amino acid modification patterns to identify a novel role for Rpd3 and Ume6, two components of a histone deacetylase complex already known to repress early meiosis-specific genes in dividing cells, in mitotic repression of meiosis-specific transcript isoforms. Our findings classify developmental stage-specific early, middle and late meiotic transcript isoforms, and they point to a novel HDAC-dependent control mechanism for flexible transcript architecture during cell growth and differentiation. Since Rpd3 is highly conserved and ubiquitously expressed in many tissues, our results are likely relevant for development and disease in higher eukaryotes., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Differentiation programs require strict spatial and temporal control of gene transcription. Genes expressed during meiotic development in Saccharomyces cerevisiae display transient induction and repression. Early meiotic gene (EMG) repression during mitosis is achieved by recruiting both histone deacetylase and chromatin remodeling complexes to their promoters by the zinc cluster DNA binding protein Ume6p. Ume6p repression is relieved by ubiquitin-mediated destruction that is stimulated by Gcn5p-induced acetylation. In this report, we demonstrate that Gcn5p acetylation of separate lysines within the zinc cluster domain negatively impacts Ume6p DNA binding. Mimicking lysine acetylation using glutamine substitution mutations decreased Ume6p binding efficiency and resulted in partial derepression of Ume6p-regulated genes. Consistent with this result, molecular modeling predicted that these lysine side chains are adjacent to the DNA phosphate backbone, suggesting that acetylation inhibits Ume6p binding by electrostatic repulsion. Preventing acetylation did not impact final EMG induction levels during meiosis. However, a delay in EMG induction was observed, which became more severe in later expression classes, ultimately resulting in delayed and reduced execution of the meiotic nuclear divisions. These results indicate that Ume6p acetylation ensures the proper timing of the transient transcription program during meiotic development.

Previous kinetic investigations of the N-terminal RNA Recognition Motif (RRM) domain of spliceosomal A protein of the U1 small nuclear ribonucleoprotein particle (U1A) interacting with its RNA target U1 hairpin II (U1hpII) provided experimental evidence for a 'lure and lock' model of binding. The final step of locking has been proposed to involve conformational changes in an α-helix immediately C-terminal to the RRM domain (helix C), which occludes the RNA binding surface in the unbound protein. Helix C must shift its position to accommodate RNA binding in the RNA-protein complex. This results in a new hydrophobic core, an intraprotein hydrogen bond and a quadruple stacking interaction between U1A and U1hpII. Here, we used a surface plasmon resonance-based biosensor to gain mechanistic insight into the role of helix C in mediating the interaction with U1hpII. Truncation, removal or disruption of the helix exposes the RNA-binding surface, resulting in an increase in the association rate, while simultaneously reducing the ability of the complex to lock, reflected in a loss of complex stability. Disruption of the quadruple stacking interaction has minor kinetic effects when compared with removal of the intraprotein hydrogen bonds. These data provide new insights into the mechanism whereby sequences C-terminal to an RRM can influence RNA binding.

Ume6p represses early meiotic gene transcription in Saccharomyces cerevisiae by recruiting the Rpd3p histone deacetylase and chromatin-remodeling proteins. Ume6p repression is relieved in a two-step destruction process mediated by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. The first step induces partial Ume6p degradation when vegetative cells shift from glucose- to acetate-based medium. Complete proteolysis happens only upon meiotic entry. Here we demonstrate that the first step in Ume6p destruction is controlled by its acetylation and deacetylation by the Gcn5p acetyltransferase and Rpd3p, respectively. Ume6p acetylation occurs in medium lacking dextrose and results in a partial destruction of the repressor. Preventing acetylation delays Ume6p meiotic destruction and retards both the transient transcription program and execution of the meiotic nuclear divisions. Conversely, mimicking acetylation induces partial destruction of Ume6p in dextrose medium and accelerates meiotic degradation by the APC/C. These studies reveal a new mechanism by which acetyltransferase activity induces gene expression through targeted destruction of a transcriptional repressor. These findings also demonstrate an important role for nonhistone acetylation in the transition between mitotic and meiotic cell division.

The yeast cyclin-C-Cdk8p kinase complex represses the transcription of a subset of genes involved in the stress response. To relieve this repression, cyclin C is destroyed in cells exposed to H(2)O(2) by the 26S proteasome. This report identifies Not4p as the ubiquitin ligase mediating H(2)O(2)-induced cyclin C destruction. Not4p is required for H(2)O(2)-induced cyclin C destruction in vivo and polyubiquitylates cyclin C in vitro by utilizing Lys48, a ubiquitin linkage associated with directing substrates to the 26S proteasome. Before its degradation, cyclin C, but not Cdk8p, translocates from the nucleus to the cytoplasm. This translocation requires both the cell-wall-integrity MAPK module and phospholipase C, and these signaling pathways are also required for cyclin C destruction. In addition, blocking cytoplasmic translocation slows the mRNA induction kinetics of two stress response genes repressed by cyclin C. Finally, a cyclin C derivative restricted to the cytoplasm is still subject to Not4p-dependent destruction, indicating that the degradation signal does not occur in the nucleus. These results identify a stress-induced proteolytic pathway regulating cyclin C that requires nuclear to cytoplasmic relocalization and Not4p-mediated ubiquitylation.

The stress-linked version of the immunocompetence handicap hypothesis has been proposed to account for inconsistencies in relationships between testosterone and immune response. The model has received some support from studies demonstrating roles of stress hormones in relationships between testosterone, immune function and secondary sexual ornamentation. Such work, however, has relied on artificial elevation of testosterone so may not reflect relationships in natural populations. We created human male facial stimuli on the basis of naturally co-occurring levels of salivary testosterone and the stress hormone cortisol. In Study 1 we tested female preferences for male faces with cues to combinations of the hormones across the menstrual cycle, and in Study 2 we tested perceptions of health and dominance in a novel set of facial stimuli. Females preferred cues to low cortisol, a preference that was strongest during the fertile phase of the menstrual cycle. The effects of cortisol on attractiveness and perceived health and dominance were contingent upon level of testosterone: the effects of the stress hormone were reduced when testosterone was high. We propose explanations for our results, including low cortisol as a cue to a heritable component of health, attractiveness as a predictor of low social-evaluative threat (and, therefore, low baseline cortisol) and testosterone as a proxy of male ability to cope efficiently with stressors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Accurate read-out of chromatin modifications is essential for eukaryotic life. Mutations in the gene encoding X-linked ATRX protein cause a mental-retardation syndrome, whereas wild-type ATRX protein targets pericentric and telomeric heterochromatin for deposition of the histone variant H3.3 by means of a largely unknown mechanism. Here we show that the ADD domain of ATRX, in which most syndrome-causing mutations occur, engages the N-terminal tail of histone H3 through two rigidly oriented binding pockets, one for unmodified Lys4 and the other for di- or trimethylated Lys9. In vivo experiments show this combinatorial readout is required for ATRX localization, with recruitment enhanced by a third interaction through heterochromatin protein-1 (HP1) that also recognizes trimethylated Lys9. The cooperation of ATRX ADD domain and HP1 in chromatin recruitment results in a tripartite interaction that may span neighboring nucleosomes and illustrates how the 'histone-code' is interpreted by a combination of multivalent effector-chromatin interactions.

The stress-linked immunocompetence handicap hypothesis (SL-ICHH) of sexual selection incorporates a role of the stress hormone corticosterone (C; cortisol in humans) in relationships between testosterone (T), immunity and secondary sexual trait expression. In support of this, C has been shown to mediate and moderate relationships between T and immune response and to be inversely related to attractiveness in some avian species. We predicted that female preferences for cues to T in human male faces would be contingent upon co-occurring cortisol levels. In study 1, we tested relationships between T and cortisol and attractiveness, masculinity and health ratings of raw male faces. We found cortisol to be inversely related to attractiveness. In study 2, we tested female preferences for male faces that were parametrically manipulated on the basis of cues to naturally co-occurring levels of T and cortisol across the menstrual cycle. Women preferred cues to low cortisol in general and in the fertile phase of the cycle, and there was an interaction between T and cortisol in general and in the non-fertile phase. Results were consistent with the SL-ICHH but not the original immunocompetence handicap model: females expressed preferences for cues to cortisol but not for cues to T, except in interaction with the stress hormone. Results inform the SL-ICHH by demonstrating female preferences for low cortisol and the nature of its interaction with T in humans, as well as indicating the traits that may be signalled by different combinations of the hormones including immune response, current health and resource acquisition characteristics.

Meiotic genes in budding yeast are repressed during vegetative growth but are transiently induced during specific stages of meiosis. Sin3p represses the early meiotic gene (EMG) by bridging the DNA binding protein Ume6p to the histone deacetylase Rpd3p. Sin3p contains four paired amphipathic helix (PAH) domains, one of which (PAH3) is required for repressing several genes expressed during mitotic cell division. This report examines the roles of the PAH domains in mediating EMG repression during mitotic cell division and following meiotic induction. PAH2 and PAH3 are required for mitotic EMG repression, while electrophoretic mobility shift assays indicate that only PAH2 is required for stable Ume6p-promoter interaction. Unlike mitotic repression, reestablishing EMG repression following transient meiotic induction requires PAH3 and PAH4. In addition, the role of Sin3p in reestablishing repression is expanded to include additional loci that it does not control during vegetative growth. These findings indicate that mitotic and postinduction EMG repressions are mediated by two separate systems that utilize different Sin3p domains.

ATRX is an X-linked gene of the SWI/SNF family, mutations in which cause syndromal mental retardation and downregulation of α-globin expression. Here we show that ATRX binds to tandem repeat (TR) sequences in both telomeres and euchromatin. Genes associated with these TRs can be dysregulated when ATRX is mutated, and the change in expression is determined by the size of the TR, producing skewed allelic expression. This reveals the characteristics of the affected genes, explains the variable phenotypes seen with identical ATRX mutations, and illustrates a new mechanism underlying variable penetrance. Many of the TRs are G rich and predicted to form non-B DNA structures (including G-quadruplex) in vivo. We show that ATRX binds G-quadruplex structures in vitro, suggesting a mechanism by which ATRX may play a role in various nuclear processes and how this is perturbed when ATRX is mutated., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The prevailing view on the effects of spinal cord injury (SCI) on emotion is that it dampens emotional experience due to a loss of peripheral bodily feedback, with the higher the lesion on the spinal cord the greater the reduction in the intensity of emotional experience. This view persists despite many studies showing an absence of such an emotional impairment in people with SCI. This study specifically aimed to investigate whether total cervical-6 spinal cord transection (i) reduces emotional expressivity and emotional awareness (ii) impairs memory for emotional material. The study contained three groups: 24 patients with SCI, 20 orthopaedic injury control (OIC) patients and 20 young adult controls. A mixed factor design was employed to examine between group and within subject differences. Participants completed the Levels of Emotional Awareness Scale (LEAS), the Berkeley Expressivity Questionnaire (BEQ), and viewed an emotionally arousing slide presentation. Thirty minutes post viewing, participants completed memory tests for the presentation. SCI patients reported greater present levels of emotional expressivity compared with perceived levels prior to their injuries. SCI and OIC groups did not differ on any of the emotional awareness variables. There was also no evidence that SCI leads to impairment in memory for emotional events. This study's findings contradict the mainstream view in the cognitive neuroscience of emotion that SCI dampens emotional experience.

The N-terminal RNA Recognition Motif (RRM1) of the spliceosomal protein U1A interacting with its target U1 hairpin II (U1hpII) has been used as a paradigm for RRM-containing proteins interacting with their RNA targets. U1A binds to U1hpII via direct interactions with a 7-nucleotide (nt) consensus binding sequence at the 5' end of a 10-nt loop, and via hydrogen bonds with the closing C-G base pair at the top of the RNA stem. Using surface plasmon resonance (Biacore), we have examined the role of structural features of U1hpII in binding to U1A RRM1. Mutational analysis of the closing base pair suggests it plays a minor role in binding and mainly prevents "breathing" of the loop. Lengthening the stem and nontarget part of the loop suggests that the increased negative charge of the RNA might slightly aid association. However, this is offset by an increase in dissociation, which may be caused by attraction of the RRM to nontarget parts of the RNA. Studies of a single stranded target and RNAs with untethered loops indicate that structure is not very relevant for association but is important for complex stability. In particular, breaking the link between the stem and the 5' side of the loop greatly increases complex dissociation, presumably by hindering simultaneous contacts between the RRM and stem and loop nucleotides. While binding of U1A to a single stranded target is much weaker than to U1hpII, it occurs with nanomolar affinity, supporting recent evidence that binding of unstructured RNA by U1A has physiological significance.

Although many accounts of facial attractiveness propose that femininity in women's faces indicates high levels of oestrogen, there is little empirical evidence in support of this assumption. Here, we used assays for urinary metabolites of oestrogen (oestrone-3-glucuronide, E1G) and progesterone (pregnanediol-3-glucuronide, P3G) to investigate the relationship between circulating gonadal hormones and ratings of the femininity, attractiveness and apparent health of women's faces. Positive correlations were observed between late follicular oestrogen and ratings of femininity, attractiveness and health. Positive correlations of luteal progesterone and health and attractiveness ratings were marginally significant. Ratings of facial attributions did not relate to hormone levels for women wearing make-up when photographed. There was no effect of sex of rater on the relationships between oestrogen and ratings of facial appearance. These findings demonstrate that female facial appearance holds detectable cues to reproductive health that are considered attractive by other people.

Previous kinetic investigations of the N-terminal RNA recognition motif (RRM) domain of spliceosomal protein U1A, interacting with its RNA target U1 hairpin II, provided experimental evidence for a 'lure and lock' model of binding in which electrostatic interactions first guide the RNA to the protein, and close range interactions then lock the two molecules together. To further investigate the 'lure' step, here we examined the electrostatic roles of two sets of positively charged amino acids in U1A that do not make hydrogen bonds to the RNA: Lys20, Lys22 and Lys23 close to the RNA-binding site, and Arg7, Lys60 and Arg70, located on 'top' of the RRM domain, away from the RNA. Surface plasmon resonance-based kinetic studies, supplemented with salt dependence experiments and molecular dynamics simulation, indicate that Lys20 predominantly plays a role in association, while nearby residues Lys22 and Lys23 appear to be at least as important for complex stability. In contrast, kinetic analyses of residues away from the RNA indicate that they have a minimal effect on association and stability. Thus, well-positioned positively charged residues can be important for both initial complex formation and complex maintenance, illustrating the multiple roles of electrostatic interactions in protein-RNA complexes.

Previous research has linked testosterone levels with sex-specific personality traits within women. The present study investigates the relation between salivary testosterone levels and specifically maternal personality traits in healthy adult women. Twenty-seven young women completed the Bem Sex Role Inventory (BSRI). Additional questions were asked about maternal personality (importance of having children, self-rated maternal/broodiness), reproductive ambition (ideal number of children, ideal own age at first child) and career orientation (importance of having career). Higher circulating testosterone levels were associated with lower scores on measures of maternal personality and reproductive ambition. There was no relation of career orientation with testosterone. A median split on BSRI masculinity revealed high scorers had higher testosterone levels than low scorers. There was no relation of BSRI femininity with testosterone. Results suggest maternal tendencies may be partly androgen driven.

Previous studies of changes in women's behavior during the menstrual cycle have offered insight into the motivations underpinning women's preferences for social cues associated with possible direct benefits (e.g., investment, low risk of infection) and indirect benefits (e.g., offspring viability). Here we sought to extend this work by testing for systematic variation in women's preferences for male and female faces and in their attitudes to their romantic relationship during the menstrual cycle. In Study 1, we found partnered women's reported commitment to their romantic relationship and preferences for femininity in male and female faces were strongest on days of the menstrual cycle when progesterone levels are increased (and fertility is low). Happiness in relationships did not change across the cycle. In Study 2, we found that the effect of cycle phase on women's preference for feminine faces was independent of increased attraction to apparent health in faces during the luteal phase. Collectively, these findings are further evidence that women's preferences for social cues associated with possible direct benefits and commitment to relationships are strongest during conditions characterized by raised progesterone level, while attraction to men displaying cues associated with possible indirect benefits is strongest when women are most fertile.

The A protein of the U1 small nuclear ribonucleoprotein particle, interacting with its stem-loop RNA target (U1hpII), is frequently used as a paradigm for RNA binding by recognition motif domains (RRMs). U1A/U1hpII complex formation has been proposed to consist of at least two steps: electrostatically mediated alignment of both molecules followed by locking into place, based on the establishment of close-range interactions. The sequence of events between alignment and locking remains obscure. Here we examine the roles of three critical residues, Tyr13, Phe56 and Gln54, in complex formation and stability using Biacore. Our mutational and kinetic data suggest that Tyr13 plays a more important role than Phe56 in complex formation. Mutational analysis of Gln54, combined with molecular dynamics studies, points to Arg52 as another key residue in association. Based on our data and previous structural and modeling studies, we propose that electrostatic alignment of the molecules is followed by hydrogen bond formation between the RNA and Arg52, and the sequential establishment of interactions with loop bases (including Tyr13). A quadruple stack, sandwiching two bases between Phe56 and Asp92, would occur last and coincide with the rearrangement of a C-terminal helix that partially occludes the RRM surface in the free protein.

Neuronal cultures in vitro readily oxidized both D-[(14)C]glucose and l-[(14)C]lactate to (14)CO(2), whereas astroglial cultures oxidized both substrates sparingly and metabolized glucose predominantly to lactate and released it into the medium. [(14)C]Glucose oxidation to (14)CO(2) varied inversely with unlabeled lactate concentration in the medium, particularly in neurons, and increased progressively with decreasing lactate concentration. Adding unlabeled glucose to the medium inhibited [(14)C]lactate oxidation to (14)CO(2) only in astroglia but not in neurons, indicating a kinetic preference in neurons for oxidation of extracellular lactate over intracellular pyruvatelactate produced by glycolysis. Protein kinase-catalyzed phosphorylation inactivates pyruvate dehydrogenase (PDH), which regulates pyruvate entry into the tricarboxylic acid cycle. Dichloroacetate inhibits this kinase, thus enhancing PDH activity. In vitro dichloroacetate stimulated glucose and lactate oxidation to CO(2) and reduced lactate release mainly in astroglia, indicating that limitations in glucose and lactate oxidation by astroglia may be due to a greater balance of PDH toward the inactive form. To assess the significance of astroglial export of lactate to neurons in vivo, we attempted to diminish this traffic in rats by administering dichloroacetate (50 mgkg) intravenously to stimulate astroglial lactate oxidation and then examined the effects on baseline and functionally activated local cerebral glucose utilization (lCMR(glc)). Dichloroacetate raised baseline lCMR(glc) throughout the brain and decreased the percent increases in lCMR(glc) evoked by functional activation. These studies provide evidence in support of the compartmentalization of glucose metabolism between astroglia and neurons but indicate that the compartmentalization may be neither complete nor entirely obligatory.

Stimulation of astroglial glucose utilization by the Na(+)/H(+) exchanger monensin is only partially blocked by ouabain. The present studies show that monensin also raises intracellular pH in astroglia. Because increased pH stimulates phosphofructokinase activity, the ouabain-insensitive portion of the stimulation of cerebral glucose utilization (CMR(glc)) appears to be due to stimulation of glycolysis by intracellular alkalinization.

2-Deoxy[14C]glucose-6-phosphate (2-[14C]DG-6-P) dephosphorylation and glucose-6-phosphatase (G-6-Pase) activity were examined in cultured rat astrocytes under conditions similar to those generally used in assays of glucose utilization. Astrocytes were loaded with 2-[14C]DG-6-P by preincubation for 15 min in medium containing 2 mM glucose and 50 microM 2-deoxy[14C]glucose (2-[14C]DG). The medium was then replaced with identical medium including 2 mM glucose but lacking 2-[14C]DG, and incubation was resumed for 5 min to diminish residual free 2-[14C]DG levels in the cells by either efflux or phosphorylation. The medium was again replaced with fresh 2-[14C]DG-free medium, and the incubation was continued for 5, 15, or 30 min. Intracellular and extracellular 14C contents were measured at each time point, and the distribution of 14C between 2-[14C]DG and 2-[14C]DG-6-P was characterized by paper chromatography. The results showed little if any hydrolysis of 2-[14C]DG-6-P or export of free 2-[14C]DG from cells to medium; there were slightly increasing losses of 2-[14C]DG and 2-[14C]DG-6-P into the medium with increasing incubation time, but they were in the same proportions found in the cells, suggesting they were derived from nonadherent or broken cells. Experiments carried out with medium lacking glucose during the assay for 2-deoxyglucose-6-phosphatase activity yielded similar results. Evidence for G-6-Pase activity was also sought by following the selective detritiation of glucose from the 2-C position when astrocytes were incubated with [2-3H]glucose and [U-14C]glucose in the medium. No change in the 3H/14C ratio was found in incubations for as long as 15 min. These results indicate negligible G-6-Pase activity in cultured astrocytes.

Studies of local glucose utilization in neural tissues in vivo with the autoradiographic [14C]deoxyglucose method have demonstrated that energy metabolism increases almost linearly with the degree of functional activation, i.e. spike frequency, in the terminal projection zones of activated pathways. The increased metabolism is found in neuropil and is minimal or undetectable in neuronal cell bodies. Electrical stimulation, increased extracellular [K+] ([K+]o), or opening of Na+ channels with veratridine stimulates metabolism in neutral tissues, and this increase is blocked by ouabain, a specific inhibitor of Na+,K(+)-ATPase. Activation of this enzyme to restore ionic gradients across cellular membranes appears to mediate the function-related increase in energy metabolism. The metabolic activation is, therefore, not directly related to the functional activity itself but to processes operating to recover from that activity. The limited spatial resolution of the [14C]DG method precludes identification of cellular elements in neuropil participating in the metabolic activation, e.g. axonal terminals, dendrites, or astrocytic processes enveloping the synapses. We have, therefore, attempted to stimulate in vitro conditions to be expected from functional activation and increased spike activity in vivo, e.g. increased extracellular [K+], intracellular [Na+], or extracellular neurotransmitter levels, and examined their effects on glucose metabolism in neurons and astroglia in culture. Increased [K+]o stimulated [14C]DG phosphorylation in neuronal and mixed neuronal-astroglial cultures, but not in astroglial cultures assayed in bicarbonate buffer; it did occasionally stimulate metabolism in astroglia when assayed in HEPES or phosphate buffers, but these effects were variable and inconsistent. Veratridine (75 microM) stimulated [14C]DG phosphorylation in neurons and astroglia; these stimulations were blocked by 1 mM ouabain or 10 microM tetrodotoxin (TTX), which blocks voltage-dependent Na+ channels. The Na+ ionophore monensin (10 microM) doubled the rate of metabolism, a stimulation that was only partially blocked by ouabain and unaffected by TTX. L-Glutamate (500 microM) stimulated [14C]DG phosphorylation in astroglia, but this stimulation was probably secondary to Na+ uptake into the cells via a sodium/glutamate co-transporter because it was not blocked by inhibitors of NMDA or non-NMDA receptors but was absent in Na(+)-free medium. These results indicate that astroglia contribute to the increased energy metabolism in neuropil during functional activation by mechanisms that promote Na+ entry into the cells.

Effects of increasing extracellular K+ or intracellular Na+ concentrations on glucose metabolism in cultures of rat astroglia and neurons were examined. Cells were incubated in bicarbonate buffer, pH 7.2, containing 2 mM glucose, tracer amounts of [14C]deoxyglucose ([14C]dGlc), and 5.4, 28, or 56 mM KCl for 10, 15, or 30 min, and then for 5 min in [14C]dGlc-free buffer to allow efflux of unmetabolized [14C]dGlc. Cells were then digested and assayed for labeled products, which were shown to consist of 96-98% [14C]deoxyglucose 6-phosphate. Increased K+ concentrations significantly raised [14C]deoxyglucose 6-phosphate accumulation in both neuronal and mixed neuronal-astroglial cultures at 15 and 30 min but did not raise it in astroglial cultures. Veratridine (75 microM), which opens voltage-dependent Na+ channels, significantly raised rates of [14C]dGlc phosphorylation in astroglial cultures (+20%), and these elevations were blocked by either 1 mM ouabain, a specific inhibitor of Na+,K(+)-ATPase (EC 3.6.1.37), or 10 microM tetrodotoxin, which blocks Na+ channels. The carboxylic sodium ionophore, monensin (10 microM), more than doubled [14C]dGlc phosphorylation; this effect was only partially blocked by ouabain and unaffected by tetrodotoxin. L-Glutamate (500 microM) also stimulated [14C]dGlc phosphorylation in astroglia--not through N-methyl-D-aspartate or non-N-methyl-D-aspartate receptor mechanisms but via a Na(+)-dependent glutamate-uptake system. These results indicate that increased uptake of Na+ can stimulate energy metabolism in astroglial cells.

Changing the medium of primary cell cultures of CNS origin causes severe damage that is mediated via the N-methyl-D-aspartate (NMDA)-type of glutamate receptors and dependent on the presence of glutamine in the medium. Data presented here show that glutamine has two roles in culture damage: glutamine is contaminated with a small amount of glutamate, which is responsible for initiating culture damage, and glutamine is the source of the glutamate that is produced extracellularly in damaged cultures. The NMDA receptor plays a critical role minutes after medium change when the glutamate contaminating the glutamine binds to NMDA receptors; during this time, addition of a low level (10-20 microM) of 2-amino-5-phosphonovaleric acid can block most culture damage and the appearance of extracellular glutamate. A higher level (300 microM) of 2-amino-5-phosphonovaleric acid can protect cultures when added at much later times (30-60 min). Between 3 and 6 h after medium change, the concentration of extracellular glutamate starts to rise and accumulates until the end of the culture period (20 h). Medium removed from cultures at 3 h or later after medium change and incubated alone (i.e., with no cells) also continues to generate glutamate; filtration (0.22 microns pore size) or centrifugation (18,000 g) stops the appearance of this glutamate. 6-Diazo-5-oxo-L-norleucine, an inhibitor of the mitochondrial enzyme glutaminase, blocks the generation of glutamate. Mitochondria or mitochondrial fragments are probably released from the damaged cells and then convert extracellular glutamine to glutamate, resulting in generation of a high extracellular glutamate concentration.

Dopaminergic neurons from embryonic rat mesencephalon were grown in simple serum-free media. The cells develop over a period of several weeks in vitro, particularly between day 14 and day 23. Removing the culture medium and replacing it with fresh medium during this interval caused severe damage to the cultures; this damage is mediated by excitatory amino acids acting through glutamate receptors. Damage could be completely prevented by antagonists of the N-methyl-D-aspartate subtype of glutamate receptor. As expected, medium that contains glutamate (i.e., Ham's F-12 medium) caused damage; however, medium that contains no glutamate or aspartate (i.e., Dulbecco's modified Eagle medium) also caused severe damage, and most of the damage was dependent on the presence of glutamine in the medium. The presence of the antibiotics penicillin and streptomycin greatly enhanced damage caused by medium change.

Myelin basic protein of rabbit brain (Mr = 18,200) was initially freed of the bulk of the nonphosphorylated species (mainly component 1) by Cm-cellulose chromatography at high pH. The remainder of the protein was subjected to peptic digestion at pH 6.00, which resulted in specific, essentially complete cleavage at several bonds (Phe-44--Phe-45, Phe-87--Phe-88, Leu-109--Ser-110, and Leu-151--Phe-152) and partial cleavage at the Tyr-14--Leu-15 bond. Gel filtration of the digest through Sephadex G-25 (fine) yielded three fractions, the first containing primarily peptides 1-44 and 45-87, the second peptides 15-44, 88-109, and 110-151, and the third peptides 1-14 and 152-168. Each fraction was chromatographed on Cm-cellulose at pH 8.2, and the resulting subfractions and partially purified peptides were analyzed for phosphoserine and phosphothreonine. Materials containing significant amounts of the phosphoamino acids were subsequently chromatographed on Cm-cellulose at pH 4.65, and the analyses for phosphoserine and phosphothreonine were repeated. The resulting purified peptic phosphopeptides were identified by amino acid analysis and tryptic peptide mapping. Comparison of the maps with those of the unphosphorylated counterparts located the tryptic phosphopeptides. These were recovered and their identities were established by amino acid analysis. In those cases where the phosphopeptide contained 2 Ser residues, the position of the phosphoserine was established by aminopeptidase M digestion. Five phosphorylation sites were found: Ser-7, Ser-56, Thr-96, Ser-113, and Ser-163. Only a small fraction of these sites was phosphorylated in the total basic protein, with values ranging from about 2 (ser-113) to 6% (Thr-96). With the possible exception of Ser-56, these sites are not the ones that have been reported to be phosphorylated in vitro by cyclic AMP-dependent protein kinase.

Rabbit myelin basic protein (BP) was subjected to partial cleavage with plasmin, and 15 cleavage products were isolated by a combination of gel filtration and ion-exchange chromatography. Their identification was achieved by amino acid analysis and tryptic peptide mapping, supplemented in some instances by carboxy-terminal analyses with carboxypeptidases A, B, and Y and amino-terminal analyses with dipeptidyl aminopeptidase I. The results showed that major plasmic cleavage sites included the Lys89-Asn90, Lys133-Ser134, and Lys153-Leu154 bonds. Cleavages also occurred at the Arg31-His32, Lys53-Arg54, and Arg25-His26 bonds, but these appeared to be less extensive. A large number of additional peptides were produced in relatively low yield. The smaller of these were isolated from heterogeneous fractions by high-voltage electrophoresis-TLC. Amino acid analysis of these peptides showed that minor cleavage sites included the Arg9-His10, Lys13-Tyr14, Lys103-Gly104, Lys137-Gly138, Lys140-Gly141, and Arg160-Ser161 bonds. In spite of a lower selectivity toward peptide bonds in BP as compared with pepsin, cathepsin D, and thrombin, plasmin has the advantage over the former proteinases in that it does not cleave at or near the Phe44-Phe45 bond. Instead it cleaves at the Arg31-His32 and Lys53-Arg54 bonds, thus preserving the entire hydrophobic sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe as well as short sequences to either side.

Rabbit myelin basic protein (BP) contains several Arg-X bonds with differing susceptibilities to thrombic cleavage as measured by the yields of the various cleavage products obtained under three different conditions. Under conditions where the thrombin-to-substrate ratio was very low (1 NIH unit/mg BP), the concentration of substrate was relatively low (4 mg BP/ml), and the incubation time was short (2 h), the rabbit BP was cleaved essentially completely and specifically at a single site, the Arg(95)-Thr(96) bond. The BPs of other species (beef, pig, guinea pig, rat) were similarly cleaved, no doubt because all have the same amino acid sequence in this region of the protein. Under conditions in which the enzyme-to-substrate ratio and the substrate concentration were higher (2 NIH units/mg BP, 8 mg BP/ml) and the incubation time was long (24 h), additional, partial cleavages occurred, principally at the Arg(43)-Phe(44) and Arg(128)-Ala(129) bonds, but with some cleavage at the Arg(31)-His(32) and Arg(63)-Thr(64) bonds as well. Under conditions in which all three variables were elevated (5 NIH units/mg peptide, 20 mg peptide/ml, 24 h), more extensive cleavage occurred at the above sites. In peptide (96-168), which we examined in detail, nearly complete cleavage of the Arg(128)-Ala(129) bond occurred, with partial cleavage at the unmethylated Arg(105)-Gly(106), Arg(111)-Phe(112), Arg(150)-Leu(151), and Arg(160)-Ser(161) bonds. The susceptibilities to cleavage of the Arg-X bonds in the BP can be explained with varying degrees of success in terms of the known specificity of thrombin. Cleavage of two of the bonds, Arg(128)-Ala(129) and Arg(160)-Ser(161), suggests the occurrence of a chain reversal or beta-turn in the sequence preceding the scissile bonds. Most cleavages of the BP with thrombin do not occur in the more hydrophobic regions; in particular, the hydrophobic region in the center of the molecule that includes the Phe-Phe(87-88) sequence is left intact.

The specificities of two monoclonal IgM antibodies (18.25 and 21.14.2) evoked in mice with guinea pig myelin basic protein were examined and interpreted in terms of a specific folding of the protein's polypeptide chain. Studies with guinea pig and rabbit myelin basic protein fragments showed that a region encompassing the central Phe-Phe (87-88) sequence is obligatory, but not sufficient, for reactivity with antibody 18.25. Appreciable reactivity was observed for rabbit peptides 22-95 and 45-151, and lower, but significant, reactivity was shown by peptide 32-95. Only very weak reactivity was seen with peptide 44-95. No reactivity was observed with peptide 1-95 after its lysine residues were acetylated, acetamidinated, or guanidinated. These results have been interpreted in terms of a polypeptide chain folding that creates an epitope within sequence Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val (84-92). The specific conformation of this epitope, which includes probably the Lys-89 and possibly the Asn-90 and Val-92 side chains, could be formed by the association of sequence 84-92 with either sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe (37-45) or with sequence Val-Leu-Ser-Arg-Phe (108-112) to form beta-sheet structures essentially identical with those that appear to be present in the intact BP [Martenson R.E.J. Neurochem. 46, 1612-1622 (1986)]. The second monoclonal antibody, no. 21.14.2, reacts only with guinea pig myelin basic protein and fragments containing the species-restricted sequence Arg-Ala-Asp-Tyr-Lys-Ser-Lys (129-135).(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of rabbit myelin basic protein component 1 with pepsin (enzyme:substrate, 1:500 w/w) in 0.5 M-ammonium formate (pH 6.00) for 15-20 min at room temperature resulted in limited cleavage of the protein. The resulting fragments were isolated by ion-exchange chromatography and gel filtration and identified by amino acid and COOH-terminal analyses and by tryptic peptide mapping. All of the possible products resulting from incomplete cleavages at the highly susceptible Phe44-Phe45, Phe87-Phe88, Leu109-Ser110, and Leu151-Phe152 bonds were isolated: peptides (1-151), (1-109), (1-87), (45-168), (45-151), (45-109), (88-168), (88-151), and (110-168). Of these, peptides (1-151), (1-87), and (88-151) were recovered in the greatest yield (0.14-0.19 mol per mol of starting protein). Relatively low yields (0.04 mol/mol starting protein) were obtained for peptides (1-109) and (110-168), indicating that the Leu109-Ser110 bond is somewhat more resistant to peptic cleavage than are the Phe-Phe and Leu-Phe bonds. Smaller fragments of the basic protein were also recovered: peptides (1-44), (1-28), (45-87), (88-109), (110-151), and (152-168). Many of the individual peptides could be readily identified in electrophoretograms of the total peptic digest. The relative electrophoretic mobilities of the above-mentioned peptides, together with the previously isolated peptides (1-14) and (15-44), were determined in 15% (w/w) polyacrylamide slab gels containing 1 M-acetic acid and 8 M-urea.