The molecular structure of (PSH+)(nido-7,8-C2B9H12-) determined by neutron diffraction (PS = proton sponge, 1,8-bis(dimethylamino)naphthalene).
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Journal Article↗
Biomedical subjects
A L Johnson
Publications and source records attributed to A L Johnson.
Rme1, which controls CLN2 expression in Saccharomyces cerevisiae, is a nuclear protein that is cell cycle regulated.
In Saccharomyces cerevisiae commitment to cell division occurs at a point in G1 termed Start. This important transition is regulated by the cyclin-dependent kinase Cdc28, in association with the G1 cyclins Cln1, 2 and 3. Transcription of the G1 cyclins is induced by the transcription factor complexes SBF (Swi4-Swi6) and MBF (Mbp1-Swi6); however, data suggest that other proteins are also able to regulate their expression. We previously identified Rme1, a transcription factor with a well documented role in negatively regulating IME1 expression and meiosis, as an activator of CLN2 transcription. We now show that Rme1 acts through two specific Rme1 response elements in the CLN2 promoter to induce expression of the gene. We have analysed in detail the timing of RME1 transcription at the end of mitosis and in G1, and the roles of the transcription factors Ace2 and Swi5 in mediating this expression. We also demonstrate that the Rme1 protein is cell cycle regulated, peaking in G1 and appearing in the nucleus at this time. Finally, the role of RME1 in cell cycle regulation is confirmed by the observation of periodic RME1 expression in diploid cells, where it has no IME1 repressor function; this finding emphasises its role in the regulation of CLN2 expression in G1.
Blotting, Northern↗
A study of the relationship between participation in common leisure activities and seizure occurrence.
OBJECTIVE: To investigate whether participation in a structured programme of fairly intensive leisure activities increased seizure occurrence. MATERIAL AND METHODS: 212 adults with medically-intractable epilepsy were closely monitored for seizure occurrence during an "activities day", i.e. a day consisting of various structured sessions of leisure pursuits (video game play, reading, word puzzles, television, physical exercise), and during other days of relative rest, whilst undergoing prolonged video EEG monitoring. RESULTS: The relative risk of seizures did not differ significantly during activities days [0.71 (95% CL: 0.38 to 1.33)] compared with days of relative rest. CONCLUSIONS: These findings fail to provide empirical support for the hypothesis that cognitive exertion has an adverse effect on seizure control.
Activities of Daily Living↗
Overlapping and distinct roles of the duplicated yeast transcription factors Ace2p and Swi5p.
The yeast transcription factors Ace2p and Swi5p regulate the expression of several target genes involved in mating type switching, exit from mitosis and cell wall function. We describe the analysis of 12 novel targets, some regulated by Ace2p or Swi5p alone and some by both. We show that Ace2p is the major regulator of four genes (CTS1, YHR143W, SCW11 and YER124C). Expression of all four is inhibited by Swi5p. Like Cts1p and Scw11p, the two new Ace2p targets are associated with cell wall metabolism. Yhr143p is localized to the cell wall, and deletion affects cell separation and enhances pseudohyphal growth. Deleting YER124C also affects cell separation and sensitivity to drugs targeted against the cell wall. Expression of PIR1, YPL158C and YNL046W is dependent on Swi5p alone. In contrast, expression of YBR158W, YNL078W and YOR264W is minimized when both ace2 and swi5 are disrupted. We propose that, although Ace2p and Swi5p co-operate to induce the expression of a subset of genes, some functional divergence has occurred. This results in a delay in the expression of those genes predominantly regulated by Ace2p, compared with those predominantly regulated by Swi5p.
Cell Cycle Proteins↗
The Spo12 protein of Saccharomyces cerevisiae: a regulator of mitotic exit whose cell cycle-dependent degradation is mediated by the anaphase-promoting complex.
The Spo12 protein plays a regulatory role in two of the most fundamental processes of biology, mitosis and meiosis, and yet its biochemical function remains elusive. In this study we concentrate on the genetic and biochemical analysis of its mitotic function. Since high-copy SPO12 is able to suppress a wide variety of mitotic exit mutants, all of which arrest with high Clb-Cdc28 activity, we speculated whether SPO12 is able to facilitate exit from mitosis when overexpressed by antagonizing mitotic kinase activity. We show, however, that Spo12 is not a potent regulator of Clb-Cdc28 activity and can function independently of either the cyclin-dependent kinase inhibitor (CDKi), Sic1, or the anaphase-promoting complex (APC) regulator, Hct1. Spo12 protein level is regulated by the APC and the protein is degraded in G1 by an Hct1-dependent mechanism. We also demonstrate that in addition to localizing to the nucleus Spo12 is a nucleolar protein. We propose a model where overexpression of Spo12 may lead to the delocalization of a small amount of Cdc14 from the nucleolus, resulting in a sufficient lowering of mitotic kinase levels to facilitate mitotic exit. Finally, site-directed mutagenesis of highly conserved residues in the Spo12 protein sequence abolishes both its mitotic suppressor activity as well as its meiotic function. This result is the first indication that Spo12 may carry out the same biochemical function in mitosis as it does in meiosis.
Amino Acid Sequence↗
Cognition and survival: an exploration in a large multicentre study of the population aged 65 years and over.
BACKGROUND: Understanding the patterns in determinants of survival becomes increasingly important as the population ages. Dementia is known to shorten survival as is impaired cognition. Whether this is a continuous phenomenon and independent of other explanatory variables is less clear. OBJECTIVES: To examine a population-based dataset in which a measure of cognitive function (Mini-Mental State Examination [MMSE]), self-reported physical health and lifestyle variables were measured at outset, with monitoring for mortality thereafter. METHODS: The five identical sites of the Medical Research Council Cognitive Function and Ageing Study (MRC CFAS) were analysed, with populations in rural Cambridgeshire, Gwynedd, Newcastle, Nottingham and Oxford. Survival curves were modelled and stratified analyses carried out, with physical disease, sociodemographic variables and lifestyle variables as covariates. RESULTS: There was a strong and consistent reduction in survival probability for each decrement in MMSE. Adjustment for known confounders did not alter this pattern. Social class and education in particular had no additional effect. Self-reported health was the only other associated variable. CONCLUSION: Cognitive function appears to be a marker of capacity for survival in the UK. Terminal decline can account for some of this. Actuarial survival provided here can give carers and service providers an idea of prognosis at given ages and levels of cognition, and provide baseline data for those planning interventions in similar groups.
Aged↗
Activation of the Akt/protein kinase B signaling pathway is associated with granulosa cell survival.
Follicles from the hen ovary that have been selected into the preovulatory hierarchy are committed to ovulation and rarely become atretic under normal physiological conditions. In part, this is attributed to the resistance of the granulosa layer to apoptosis. The present studies were conducted to evaluate the role of the phosphatidylinositol (PI) 3-kinase/Akt signaling pathway in hen granulosa cell survival and, by implication, follicle viability. Cloning of the chicken akt2 homologue revealed a high degree of amino acid homology to its mammalian counterparts within the catalytic domain, plus complete conservation of the putative Thr(308) and Ser(474) phosphorylation sites. Treatment of granulosa cells from the three largest preovulatory follicles with insulin-like growth factor (IGF)-I and, to a lesser extent, transforming growth factor (TGF)-alpha induces rapid phosphorylation of Akt, and such phosphorylation is effectively blocked by the PI 3-kinase-inhibitor LY294006. Serum withdrawal from cultured cells for 33-44 h initiates oligonucleosome formation, an indicator of apoptotic cell death, whereas cotreatment with IGF-I prevents this effect. Moreover, treatment of cultured cells for 20 h with LY294006 induces apoptosis. The potential for nonspecific cell toxicity following LY294006 treatment is considered unlikely because of the ability of either LH or 8-bromo cAMP cotreatment to block LY294006-induced cell death. Finally, both IGF-I and TGF-alpha also activate mitogen-activated protein (MAP) kinase signaling, at least in part, through the phosphorylation of ERK: However, treatment with neither U0126 nor PD98059 (inhibitors of MAP kinase kinase) induced cell death in cultured granulosa cells, despite the ability of each inhibitor to effectively block Erk phosphorylation. Taken together, these results provide evidence for a role of the Akt signaling pathway in promoting cell survival within the preovulatory follicle granulosa layer. In addition, the data indicate the importance of an alternative survival pathway mediated via gonadotropins and protein kinase A independent of Akt signaling.
Animals↗
Expression and regulation of Fas antigen and tumor necrosis factor receptor type I in hen granulosa cells.
It is now well established that vertebrate ovarian follicles undergo atresia via apoptosis, a process that is initiated within the granulosa cell layer of undifferentiated follicles. Although the exact signals, membrane-bound receptors, and associated intracellular signaling pathways leading to apoptosis within granulosa cells have yet to be established, it is evident that multiple and redundant pathways exist. Fas, together with its ligand, has been the most commonly studied death-inducer in the mammalian ovary; however, nothing is currently known regarding expression of either Fas or the related tumor necrosis factor receptor type 1 (TNFR1), in avian species. Based on characterization of a chicken fas partial cDNA, which includes the entire death domain, the deduced amino acid sequence shows 37% identity (53% positive) to human Fas. Northern blot analysis demonstrates low expression of the 2.0-kilobase fas transcript in most tissues, including the granulosa layer, and highest levels are found in the spleen, theca tissue, and the postovulatory follicle. Significantly, fas and tnfr1 mRNA levels are higher in atretic follicles than in nonatretic, prehierarchal (3- to 8-mm diameter) follicles. Moreover, both fas and tnfr1 mRNA levels are up-regulated by twofold to eightfold in granulosa cells following plating in the presence of fetal bovine serum, with the most dramatic increase found in fas expression within prehierarchal follicle granulosa. Coculture with transforming growth factor (TGF) beta attenuates this increase for both receptors, whereas cAMP attenuates only the up-regulation of fas. By comparison, treatment with TGFalpha enhances expression of tnfr1, but not fas, mRNA. Taken together, these data are the first to implicate fas as a mediator of granulosa cell apoptosis in a nonmammalian vertebrate, and to implicate the protein kinase A signaling pathway in down-regulating fas expression. In addition, data provided demonstrate the presence of multiple death domain-containing TNFR family members simultaneously expressed within hen granulosa cells, each of which may be regulated by separate signaling pathways.
Alternative Splicing↗
Post-translational processing of surfactant protein-C proprotein: targeting motifs in the NH(2)-terminal flanking domain are cleaved in late compartments.
Rat surfactant protein (SP)-C is a 3.7-kD hydrophobic lung-specific protein generated from proteolytic processing of a 21-kD propeptide (SP-C(21)). We have demonstrated that initial post-translational processing of SP-C(21) involves two cleavages of the COOH-terminus (Beers and colleagues, J. Biol. Chem. 1994;269:20,318--20,328). The goal of the current study was to define processing and function of the NH(2)-terminal flanking domain. Epitope-specific antisera directed against spatially distinct regions of the NH(2) terminus, NPROSP-C(2-9) (epitope = D(2)-L(9)) and NPROSP-C(11-23) (= E(11)-Q(23)) were produced. By Western blotting, both antisera identified SP-C(21) in microsomes. A 6-kD form (SP-C(6)), enriched in lamellar bodies (LBs), was detected only by NPROSP-C(11-23) and not extractable with NaCO(3) treatment. Immunogold staining of ultrathin lung sections with NPROSP-C(11-23) identified proSP-C in both multivesicular bodies (mvb) and LBs whereas NPROSP-C(2-9) labeled only mvb. (35)S-pulse chase analysis demonstrated synthesis of SP-C(21) and three intermediate forms (SP-C(16), SP-C(7), and SP-C(6)). Complete processing involved four separate cleavages with a precursor- product relationship between the low molecular weight forms SP-C(7) and SP-C(6). Fluorescence microscopy of A549 cells expressing fusion proteins of enhanced green fluorescent protein (EGFP) and proSP-C NH(2)-terminal deletion mutants showed targeting of EGFP/SP-C(1-194) and EGFP/SP-C(10-194) to early endosomal antigen-1-negative, CD-63-positive cytoplasmic vesicles whereas EGFP/SP-C(19-194), EGFP/SP-C(Delta 10-18), and EGFP/SP-C(24-194) were restricted to the endoplasmic reticulum (ER). We conclude that synthetic processing includes a previously unrecognized cleavage of the proximal NH(2) terminus (M(1)-L(9)), which occurs after removal of COOH-flanking domains (H(59)-I(194)) but before packaging in LBs, and that the region M(10)-T(18) is required for targeting of proSP-C to post-ER vesicular compartments in the biosynthetic pathway.
Amino Acid Sequence↗
Regulation of steroidogenic acute regulatory protein and luteinizing hormone receptor messenger ribonucleic acid in hen granulosa cells.
The regulation of steroidogenic acute regulatory protein (StAR) in vitro by gonadotropins was investigated in granulosa cells from prehierarchal and preovulatory hen follicles. Basal levels of StAR messenger RNA (mRNA) in undifferentiated granulosa cells from prehierarchal (6- to 8-mm) follicles were consistently low, but detectable, and were significantly increased by treatment with 8-bromo-cAMP and FSH (but not LH) within 3-6 h of culture. After 20 h of culture, 8-bromo-cAMP, FSH, and LH each increased StAR mRNA levels above those in control cultured cells, and the delayed response to LH treatment was associated with increased levels of LH receptor (LH-R) mRNA. On the other hand, inhibition of mitogen-activated protein (MAP) kinase signaling, using the MAP kinase kinase inhibitors U0126 and PD98059, in the presence of FSH further increased StAR mRNA and protein levels, LH-R mRNA levels, and progesterone synthesis compared with those in cells cultured with FSH alone. The highest basal expression of StAR mRNA during follicle development was found in granulosa from the largest (F1) preovulatory follicle, with comparatively lower levels in granulosa from less mature (F2 plus F3) preovulatory follicles. Treatment with LH rapidly increased StAR mRNA and protein (but not LH-R mRNA) expression in cultures of F1 granulosa and in combined F2 plus F3 granulosa within 3 h, although the magnitude of stimulation was greater in F2 plus F3 granulosa. Compared with results from granulosa cells from prehierarchal follicles cultured for 20 h, inhibition of MAP kinase signaling in the presence of LH for 1 h failed to further enhance levels of StAR or LH-R expression or progesterone production in F2 plus F3 follicle granulosa compared with the effect of LH treatment alone. These results demonstrate that StAR expression in the hen ovary is up-regulated by gonadotropins at least in part via cAMP signaling. The ability of MAP kinase kinase inhibitors to potentiate gonadotropin-induced StAR and LH-R expression plus progesterone synthesis in prehierarchal follicle granulosa cells in vitro suggests that inhibition of paracrine or autocrine factor-mediated MAP kinase signaling in vivo may be a prerequisite for the full potentiation of granulosa cell steroidogenesis that occurs after recruitment into the preovulatory hierarchy. Finally, these results fail to support a role for MAP kinase signaling in acutely modulating LH-mediated StAR expression or progesterone production in hierarchal follicles, such as occurs during the preovulatory surge of progesterone.
Animals↗
The Bub2-dependent mitotic pathway in yeast acts every cell cycle and regulates cytokinesis.
In eukaryotes an abnormal spindle activates a conserved checkpoint consisting of the MAD and BUB genes that results in mitotic arrest at metaphase. Recently, we and others identified a novel Bub2-dependent branch to this checkpoint that blocks mitotic exit. This cell-cycle arrest depends upon inhibition of the G-protein Tem1 that appears to be regulated by Bfa1/Bub2, a two-component GTPase-activating protein, and the exchange factor Lte1. Here, we find that Bub2 and Bfa1 physically associate across the entire cell cycle and bind to Tem1 during mitosis and early G1. Bfa1 is multiply phosphorylated in a cell-cycle-dependent manner with the major phosphorylation occurring in mitosis. This Bfa1 phosphorylation is Bub2-dependent. Cdc5, but not Cdc15 or Dbf2, partly controls the phosphorylation of Bfa1 and also Lte1. Following spindle checkpoint activation, the cell cycle phosphorylation of Bfa1 and Lte1 is protracted and some species are accentuated. Thus, the Bub2-dependent pathway is active every cell cycle and the effect of spindle damage is simply to protract its normal function. Indeed, function of the Bub2 pathway is also prolonged during metaphase arrests imposed by means other than checkpoint activation. In metaphase cells Bub2 is crucial to restrain downstream events such as actin ring formation, emphasising the importance of the Bub2 pathway in the regulation of cytokinesis. Our data is consistent with Bub2/Bfa1 being a rate-limiting negative regulator of downstream events during metaphase.
Anaphase-Promoting Complex-Cyclosome↗
Expression and function of brain-derived neurotrophin factor and its receptor, TrkB, in ovarian follicles from the domestic hen (Gallus gallus domesticus).
This report summarizes patterns of mRNA expression for the brain-derived neurotrophic factor (BDNF) together with its high-affinity neurotrophin receptor trkB within the hen ovary during follicle development, describes hormonal mechanisms for the regulation of trkB gene expression and provides preliminary evidence for a novel function for BDNF-mediated TrkB signaling within the granulosa layer. Levels of BDNF mRNA in the thecal layer and of trkB mRNA within the granulosa cell layer increase coincident with entrance of the follicle into the preovulatory hierarchy. Localization of the BDNF mRNA transcript correlates with expression of BDNF protein within the theca interna of preovulatory follicles, while localization of trkB mRNA and protein occurs extensively within the granulosa cell layer of preovulatory follicles. This pattern of expression suggests a paracrine relationship between theca and granulosa cells for BDNF signaling via TrkB. Vasoactive intestinal peptide and gonadotropin treatments stimulate increases in levels of trkB mRNA within cultured granulosa cells derived from both prehierarchal and preovulatory follicles, and this response is increased by co-treatment with 3-isobutyl-1-methylxanthine. Finally, BDNF treatment of cultured granulosa cells from preovulatory follicles results in a modest, but significant, reduction in basal progesterone production, whereas this effect was reversed by k252a, an inhibitor of Trk kinase activity. These results support the proposals that BDNF functions as a paracrine signal in hen granulosa cells and that its physiological functions may include the modulation of steroidogenesis.
Animals↗
Mortality in epilepsy in the first 11 to 14 years after diagnosis: multivariate analysis of a long-term, prospective, population-based cohort.
The United Kingdom National General Practice Study of Epilepsy is a prospective, population-based study of newly diagnosed epilepsy. A cohort of 792 patients has now been followed for up to 14 years (median follow-up [25th, 75th percentiles] 11.8 years, range 10.6-11.7 years), a total of 11,400 person-years. These data are sufficient for a detailed analysis of mortality in this early phase of epilepsy. Over 70% of patients in this cohort have developed lasting remission from seizures, although the mortality rate in the long term was still twice that of the general population. The standardized mortality ratio (SMR), the number of observed deaths per number of expected deaths, was 2.1 (95% confidence interval [CI] = 1.8, 2.4). Patients with acute symptomatic epilepsy (SMR 3.0; 95% CI = 2.0, 4.3), remote symptomatic epilepsy (SMR 3.7; 95% CI = 2.9, 4.6), and epilepsy due to congenital neurological deficits (SMR 25; 95% CI = 5.1, 73.1) had significantly increased long-term mortality rates, whereas patients with idiopathic epilepsy did not (SMR 1.3; 95% CI = 0.9, 1.9). This increase in mortality rate was noted particularly in the first few years after diagnosis. Multivariate Cox regression and time-dependent co-variate analyses were utilized for the first time in a prospective study of mortality in epilepsy. The former showed that patients with generalized tonic-clonic seizures had an increased risk of mortality. The hazard ratio (HR), or risk of mortality in a particular group with a particular risk factor compared to another group without that particular risk factor, was 6.2 (95% CI = 1.4, 27.7; p = 0.049). Cerebrovascular disease (HR 2.4; 95% CI = 1.7, 3.4; p < 0.0001), central nervous system tumor (HR 12.0; 95% CI = 7.9, 18.2; p < 0.0001), alcohol (HR 2.9; 95% CI = 1.5, 5.7; p = 0.004), and congenital neurological deficits (HR 10.9; 95% CI = 3.2, 36.1; p = 0.003) as causes for epilepsy and older age at index seizure (HR 1.9; 95% CI = 1.7,2.0; p < 0.0001) were also associated with significantly increased mortality rates. These hazard ratios suggest that epilepsy due to congenital neurological deficits may carry almost the same risk of mortality as epilepsy due to central nervous system tumors and that epileptic seizures subsequent to alcohol abuse may carry almost the same risk of mortality as epilepsy due to cerebrovascular disease. The occurrence of one or more seizures before the index seizure (the seizure that led to the diagnosis of epilepsy and enrolment in the study) was associated with a significantly reduced mortality rate (HR 0.57; 95% CI = 0.42, 0.76; p = 0.00001). Time-dependent co-variate analysis was used to examine the influence of ongoing factors, such as seizure recurrence, remission, and antiepileptic drug use, on mortality rates in the cohort. Seizure recurrence (HR 1.30; 95% CI = 0.84, 2.01) and antiepileptic drug treatment (HR 0.97; 95% CI = 0.67, 1.38) did not influence mortality rate. There were only 5 epilepsy-related deaths (1 each of sudden unexpected death in epilepsy, status epilepticus, burns, drowning, and cervical fracture), suggesting that death directly due to epileptic seizures is uncommon in a population-based cohort with epilepsy.
Adolescent↗
Lest we forget: Early Canadian contributions to the care of children with congenital heart malformations.
In a century of remarkable progress in medicine, one of the outstanding stories is the development of successful treatment of congenital heart malformations. This achievement is the outcome of the ideas and research of many people. Many of the early 'transforming' advances originated in Canada. Maude Abbott catalogued and classified heart malformations in a clinically meaningful manner. Arthur Charles and David Scott in Toronto, Ontario, produced a clinically useful heparin preparation, and the studies of Bill Bigelow led to the application of hypothermia in cardiac surgery. John Keith and Bill Mustard at Toronto's Hospital for Sick Children, and Arnold Johnson and Tony Dobell at the Montreal Children's Hospital, Montreal, Quebec, established the first Canadian programs devoted to the correction of congenital heart defects in childhood. Mustard devised the first widely successful operation for transposition of the great arteries. Flavio Coceani and Peter Olley discovered the role of prostaglandin E in the ductus arteriosus, and applied that knowledge clinically. The turn of the century is an appropriate time to celebrate these Canadian successes.
Canada↗
Conservation of steroidogenic acute regulatory (StAR) protein structure and expression in vertebrates.
Complementary DNAs for the open reading frames of the chicken, Xenopus and zebrafish StAR homologs were cloned along with a partial cDNA of the zebrafish homolog to MLN64, a StAR-related protein. A comparison of the amino acid sequences of piscine, amphibian, avian and mammalian StARs, indicates strong conservation of the protein across divergent vertebrate groups. On Northern blots probed with species specific StAR cDNAs, expression of StAR transcripts was observed in the ovary and adrenal of chicken, and the ovary, testis, kidney and head of zebrafish. The expression of StAR mRNA in various compartments of the hen ovary was consistent with the results of past studies on steroidogenesis; expression was first observed in follicles selected into the preovulatory hierarchy and was greatest in the largest preovulatory follicle. The expression of StAR mRNA was also consistent with aromatase expression in zebrafish ovaries. The conserved deduced protein sequence and expression pattern of StAR transcripts in chicken and zebrafish tissues, strongly suggest that StAR is also involved in the regulation of steroidogenesis in nonmammalian vertebrates.
Amino Acid Sequence↗
The forkhead protein Fkh2 is a component of the yeast cell cycle transcription factor SFF.
In the yeast Saccharomyces cerevisiae, the MADS-box protein Mcm1, which is highly related to mammalian SRF (serum response factor), forms a ternary complex with SFF (Swi five factor) to regulate the cell cycle expression of genes such as SWI5, CLB2 and ACE2. Here we show that the forkhead protein Fkh2 is a component of SFF and is essential for ternary complex formation on the SWI5 and ACE2 promoters. Fkh2 is essential for the correct cell cycle periodicity of SWI5 and CLB2 gene expression and is phosphorylated with a timing that is consistent with a role in this expression. Furthermore, investigation of the relationship between Fkh2 and a related forkhead protein Fkh1 demonstrates that these proteins act in overlapping pathways to regulate cell morphology and cell separation. This is the first example of a eukaryotic transcription factor complex containing both a MADS-box and a forkhead protein, and it has important implications for the regulation of mammalian gene expression.
Cell Cycle↗
Regulation of the Pcl7-Pho85 cyclin-cdk complex by Pho81.
Saccharomyces cerevisiae strains lacking a functional Pho85 cyclin-dependent kinase (cdk) exhibit a complex phenotype, including deregulation of phosphatase genes controlled by the transcription factor Pho4, slow growth on rich media, failure to grow using galactose, lactate or glycerol as a carbon source and hyperaccumulation of glycogen. The ability of Pho85 to regulate the transcription factor Pho4 is mediated by its association the Pho80 cyclin. Some other regulatory functions of the Pho85 cdk have been shown to be mediated via its interaction with a recently identified family of Pho80-related cyclins (Pcls). Here, we show that the poorly characterized Pho80-like protein Pcl7 forms a functional kinase complex with the Pho85 cdk, and that the activity of this complex is inhibited in response to phosphate starvation. Additionally, we show that Pcl7 interacts with the phosphate-regulated cyclin-cdk inhibitor Pho81, and that the regulation of the Pcl7-Pho85 complex in response to changes in phosphate levels is dependent on Pho81. Thus, we demonstrate for the first time that the Pho81 regulator is not dedicated to regulating Pho80, but may act to co-ordinate the activity of both the Pho80-Pho85 and Pcl7-Pho85 cyclin-cdk complexes in response to phosphate levels. We also demonstrate that expression of Pcl7 is cell cycle regulated, with maximal activity occurring in mid to late S-phase, perhaps suggesting a role for Pcl7 in cell cycle progression. Finally, we describe the phenotype of pcl7Delta and pcl6Delta yeast strains that have defects in carbon source utilization.
Amino Acid Sequence↗
The Skn7 response regulator of Saccharomyces cerevisiae interacts with Hsf1 in vivo and is required for the induction of heat shock genes by oxidative stress.
The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein-protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.
Adenosine Triphosphatases↗