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BACKGROUND Recurrent pregnancy loss, (RPL) affecting 1%–2% of couples, is defined as ≥3 consecutive pregnancy losses before 20-week' gestation. Women with RPL are routinely screened for etiological factors, but routine screening of male partners is not currently recommended. Recently it has been suggested that sperm quality is reduced in male partners of women with RPL, but the reasons underlying this lower quality are unclear. We hypothesized that these men may have underlying impairments of reproductive endocrine and metabolic function that cause reductions in sperm quality. METHODS After ethical approval, reproductive parameters were compared between healthy controls and male partners of women with RPL. Semen reactive oxygen species (ROS) were measured with a validated inhouse chemiluminescent assay. DNA fragmentation was measured with the validated Halosperm method. RESULTS Total sperm motility, progressive sperm motility, and normal morphology were all reduced in the RPL group vs controls. Mean ±SE morning serum testosterone (nmol/L) was 15% lower in RPL than in controls (controls, 19.0 ± 1.0; RPL, 16.0 ± 0.8; P < 0.05). Mean ±SE serum estradiol (pmol/L) was 16% lower in RPL than in controls (controls, 103.1 ± 5.7; RPL, 86.5 ± 3.4; P < 0.01). Serum luteinizing hormone and follicle-stimulating hormone were similar between groups. Mean ±SE ROS (RLU/sec/106 sperm) were 4-fold higher in RPL than in controls (controls, 2.0 ± 0.6; RPL, 9.1 ± 4.1; P < 0.01). Mean ±SE sperm DNA fragmentation (%) was 2-fold higher in RPL than in controls (controls, 7.3 ± 1.0; RPL, 16.4 ± 1.5; P < 0.0001). CONCLUSIONS Our data suggest that male partners of women with RPL have impaired reproductive endocrine function, increased levels of semen ROS, and sperm DNA fragmentation. Routine reproductive assessment of the male partners may be beneficial in RPL.

Gln3 activates Nitrogen Catabolite Repression, NCR-sensitive expression of the genes required for Saccharomyces cerevisiae to scavenge poor nitrogen sources from its environment. The global TorC1 kinase complex negatively regulates nuclear Gln3 localization, interacting with an α-helix in the C-terminal region of Gln3, Gln3656–666. In nitrogen replete conditions, Gln3 is sequestered in the cytoplasm, whereas when TorC1 is down-regulated, in nitrogen restrictive conditions, Gln3 migrates into the nucleus. In this work, we show that the C-terminal Gln3–Tor1 interaction site is required for wild type, rapamycin-elicited, Sit4-dependent nuclear Gln3 localization, but not for its dephosphorylation. In fact, truncated Gln31-384 can enter the nucleus in the absence of Sit4 in both repressive and derepressive growth conditions. However, Gln31-384 can only enter the nucleus if a newly discovered second positively-acting Gln3–Tor1 interaction site remains intact. Importantly, the N- and C-terminal Gln3–Tor1 interaction sites function both autonomously and collaboratively. The N-terminal Gln3–Tor1 interaction site, previously designated Gln3URS contains a predicted α-helix situated within an unstructured coiled-coil region. Eight of the thirteen serine/threonine residues in the Gln3URS are dephosphorylated 3–15-fold with three of them by 10–15-fold. Substituting phosphomimetic aspartate for serine/threonine residues in the Gln3 URS abolishes the N-terminal Gln3–Tor1 interaction, rapamycin-elicited nuclear Gln3 localization, and ½ of the derepressed levels of nuclear Gln3 localization. Cytoplasmic Gln3 sequestration in repressive conditions, however, remains intact. These findings further deconvolve the mechanisms that achieve nitrogen-responsive transcription factor regulation downstream of TorC1.

Nitrogen catabolite repression (NCR), the ability of Saccharomyces cerevisiae to use good nitrogen sources in preference to poor ones, derives from nitrogen-responsive regulation of the GATA family transcription activators Gln3 and Gat1. In nitrogen-replete conditions, the GATA factors are cytoplasmic and NCR-sensitive transcription minimal. When only poor nitrogen sources are available, Gln3 is nuclear, dramatically increasing GATA factor-mediated transcription. This regulation was originally attributed to mechanistic Tor protein kinase complex 1 (mTorC1)-mediated control of Gln3. However, we recently showed that two regulatory systems act cumulatively to maintain cytoplasmic Gln3 sequestration, only one of which is mTorC1. Present experiments demonstrate that the other previously elusive component is uncharged transfer RNA-activated, Gcn2 protein kinase-mediated general amino acid control (GAAC). Gcn2 and Gcn4 are required for NCR-sensitive nuclear Gln3-Myc13 localization, and from epistasis experiments Gcn2 appears to function upstream of Ure2. Bmh1/2 are also required for nuclear Gln3-Myc13 localization and appear to function downstream of Ure2. Overall, Gln3 phosphorylation levels decrease upon loss of Gcn2, Gcn4, or Bmh1/2. Our results add a new dimension to nitrogen-responsive GATA-factor regulation and demonstrate the cumulative participation of the mTorC1 and GAAC pathways, which respond oppositely to nitrogen availability, in the nitrogen-responsive control of catabolic gene expression in yeast.

Background Thyroid autoantibodies, specifically thyroid peroxidase antibodies, have been associated with miscarriage and pre-term birth in women with a normal thyroid function. Small randomised controlled trials have found that treatment with levothyroxine may reduce such adverse outcomes in pregnancy. Objectives The Thyroid AntiBodies and LEvoThyroxine (TABLET) trial was conducted to explore the effects of levothyroxine in euthyroid women with thyroid peroxidase antibodies. A concurrent mechanistic study was conducted to examine the effect of levothyroxine on immune responses. Design This was a randomised, double-blind, placebo-controlled, multicentre study. Setting The TABLET trial was conducted in 49 hospitals across the UK between 2011 and 2016. Participants Euthyroid women who tested positive for thyroid peroxidase antibodies, were aged between 16 and 41 years and were trying to conceive either naturally or through assisted conception were eligible. Intervention Participants were randomised to levothyroxine at a dose of 50 µg daily or placebo. The intervention was commenced preconception and continued until the end of a pregnancy. Women were given a 12-month period to conceive from randomisation. Main outcome measures The primary outcome was live birth at ≥ 34 completed weeks of gestation. The secondary outcomes included miscarriage at Methods Participants were randomised in a 1 : 1 ratio. Minimisation was implemented for age ( 2.5 mlU/l) to achieve balanced trial arms. Women were followed up every 3 months while trying to conceive to check thyroid function and general well-being, and, once pregnant, were seen each trimester: 6–8 weeks, 16–18 weeks and 28 weeks. Any abnormal thyroid results were managed in line with clinical guidance at the time. Results Of the 19,556 women screened, 1420 women were eligible and 952 were randomised to receive levothyroxine (n = 476) or placebo (n = 476). Six women from each arm either were lost to follow-up or withdrew from the trial. A total 540 women became pregnant: 266 in the levothyroxine arm and 274 in the placebo arm. The live birth rate was 37% (176/470) in the levothyroxine group and 38% (178/470) in the placebo group, translating to a relative risk of 0.97 (95% confidence interval 0.83 to 1.14; p = 0.74) and an absolute risk difference of –0.4% (95% confidence interval –6.6% to 5.8%). A subset of 49 trial participants (26 in the levothyroxine arm and 23 in the placebo arm) were recruited to assess changes in their serum chemocytokine concentrations. Treatment with levothyroxine resulted in some changes in chemocytokine concentrations in the non-pregnant state and in early pregnancy, but these had no association with clinical outcome. Conclusions Levothyroxine therapy in a dose of 50 µg per day does not improve live birth rate in euthyroid women with thyroid peroxidase antibodies. Limitations Titration of the levothyroxine dose based on thyroid-stimulating hormone/thyroid peroxidase concentrations was not explored. Future work Future research could explore the efficacy of levothyroxine administered for the treatment of subclinical hypothyroidism. Trial registration Current Controlled Trials ISRCTN15948785 and EudraCT 2011-000719-19. Funding This project was funded by the Efficacy and Mechanism Evaluation programme, a Medical Research Council and National Institute for Health Research partnership.

Background Lymphogranuloma venereum (LGV) is caused by Chlamydia trachomatis (CT) serovars L1–L3. The most recent US outbreak of LGV was in 2016 in Michigan in men who have sex with men (MSM) living with HIV. Methods To better understand LGV epidemiology in Chicago and increase provider awareness, in 2016, the Chicago Department of Public Health (CDPH) introduced a case-based reporting system for MSM with suspected LGV proctitis. Providers were asked to complete standard forms for adult MSM demonstrating symptoms of proctitis. Demographic/clinical and behavioral risk factors data were abstracted from 7/21/16 - 4/30/17. Rectal specimens found to be positive for CT on nucleic acid amplification testing were submitted for LGV laboratory confirmation. Results A total of 50 suspect LGV cases were reported to CDPH; 47 specimens were submitted to for further molecular testing: 19 were confirmed to be LGV, 10 were non-LGV/CT positive, 2 had indeterminate results and 16 were CT-negative. All confirmed cases were from rectal swabs: 21% (4/19) were non-Hispanic Black, 42% (8/19) were non-Hispanic white, 32% (6/19) were Hispanic, and 5% (1/19) were non-Hispanic Asian. The median age was 35 years (range = 21–46 years). Of 19 confirmed cases, 84% (N=16) were HIV (+), and in two cases, HIV was diagnosed at the time of LGV infection. The median CD4 count was 613 cells/ml (range = 311–1170 cells/ml, IQR=238); HIV RNA was Conclusion LGV may be suspected in MSM presenting with proctitis symptoms. These data likely underestimate the true local prevalence of LGV in Chicago since reporting was restricted to symptomatic MSM. Improvements in chlamydia case-based surveillance in key populations are critical given the association with LGV and HIV. Disclosure No significant relationships.

Background: Idiopathic recurrent pregnancy loss (RPL) is the loss of three or more consecutive pregnancies prior to 20 weeks of gestation. Female factors in RPL are well known, but the influence of sperm function on RPL has been poorly understood. Recent studies demonstrate that male partners of women with RPL have higher risk of DNA fragmentation, but the underlying reasons have not been previously studied. Determining the mechanisms of sperm damage has potential to open new therapeutic approaches for couples. Objective: To investigate the role of seminal reactive oxygen species (ROS) as a novel marker of sperm function in male partners of women with RPL. Methods: Serum levels of reproductive hormones and sperm quality of male partners with RPL (N=49) were compared with a group of unaffected controls (n=34). All participants completed a questionnaire for conditions impairing sperm quality such as testicular surgery, chronic use of medication, sexually transmitted infections, smoking and alcohol intake. Semen ROS was measured using a validated in-house chemiluminescent luminol assay. DNA fragmentation was measured using the validated Halosperm G2 kit. Results: Sperm morphology was significantly lower in the RPL group when compared with controls (% sperm with normal morphology: 4.5 ± 0.4, control; 3.4± 0.3, RPL, P0.05). Conclusions: Our data suggest that male partners of women with RPL had elevated levels of semen ROS, elevated sperm DNA fragmentation and lower percentage of normal morphology when compared with controls. Our data have important implications for the management of couples with RPL as male partners of women with RPL may benefit from routine assessment of reproductive endocrine and sperm function.

BACKGROUND: Progesterone is essential for the maintenance of pregnancy. However, whether progesterone supplementation in the first trimester of pregnancy would increase the rate of live births among women with a history of unexplained recurrent miscarriages is uncertain.METHODS: We conducted a multicenter, double-blind, placebo-controlled, randomized trial to investigate whether treatment with progesterone would increase the rates of live births and newborn survival among women with unexplained recurrent miscarriage. We randomly assigned women with recurrent miscarriages to receive twice-daily vaginal suppositories containing either 400 mg of micronized progesterone or matched placebo from a time soon after a positive urinary pregnancy test (and no later than 6 weeks of gestation) through 12 weeks of gestation. The primary outcome was live birth after 24 weeks of gestation.RESULTS: A total of 1568 women were assessed for eligibility, and 836 of these women who conceived naturally within 1 year and remained willing to participate in the trial were randomly assigned to receive either progesterone (404 women) or placebo (432 women). The follow-up rate for the primary outcome was 98.8% (826 of 836 women). In an intention-to-treat analysis, the rate of live births was 65.8% (262 of 398 women) in the progesterone group and 63.3% (271 of 428 women) in the placebo group (relative rate, 1.04; 95% confidence interval [CI], 0.94 to 1.15; rate difference, 2.5 percentage points; 95% CI, -4.0 to 9.0). There were no significant between-group differences in the rate of adverse events.CONCLUSIONS: Progesterone therapy in the first trimester of pregnancy did not result in a significantly higher rate of live births among women with a history of unexplained recurrent miscarriages. (Funded by the United Kingdom National Institute of Health Research; PROMISE Current Controlled Trials number, ISRCTN92644181.).

The TorC1 protein kinase complex is a central component in a eukaryotic cell's response to varying nitrogen availability, with kinase activity being stimulated in nitrogen excess by increased intracellular leucine. This leucine-dependent TorC1 activation requires functional Gtr1/2 and Ego1/3 complexes. Rapamycin inhibition of TorC1 elicits nuclear localization of Gln3, a GATA-family transcription activator responsible for the expression of genes encoding proteins required to transport and degrade poor nitrogen sources, e.g. proline. In nitrogen-replete conditions, Gln3 is cytoplasmic and Gln3-mediated transcription minimal, whereas in nitrogen limiting or starvation conditions, or after rapamycin treatment, Gln3 is nuclear and transcription greatly increased. Increasing evidence supports the idea that TorC1 activation may not be as central to nitrogen-responsive intracellular Gln3 localization as envisioned previously. To test this idea directly, we determined whether Gtr1/2- and Ego1/3-dependent TorC1 activation also was required for cytoplasmic Gln3 sequestration and repressed GATA factor-mediated transcription by abolishing the Gtr-Ego complex proteins. We show that Gln3 is sequestered in the cytoplasm of gtr1Δ, gtr2Δ, ego1Δ, and ego3Δ strains either long term in logarithmically glutamine-grown cells or short term after refeeding glutamine to nitrogen-limited or -starved cells; GATA factor2dependent transcription also was minimal. However, in all but a gtr1Δ, nuclear Gln3 localization in response to nitrogen limitation or starvation was adversely affected. Our data demonstrate: (i) Gtr-Ego-dependent TorC1 activation is not required for cytoplasmic Gln3 sequestration in nitrogen-rich conditions; (ii) a novel Gtr-Ego-TorC1 activation-independent mechanism sequesters Gln3 in the cytoplasm; (iii) Gtr and Ego complex proteins participate in nuclear Gln3- Myc13 localization, heretofore unrecognized functions for these proteins; and (iv) the importance of searching for new mechanisms associated with TorC1 activation and/or the regulation of Gln3 localization/function in response to changes in the cells' nitrogen environment., SCOPUS: ar.j, info:eu-repo/semantics/published

A leucine, leucyl-tRNA synthetase–dependent pathway activates TorC1 kinase and its downstream stimulation of protein synthesis, a major nitrogen consumer. We previously demonstrated, however, that control of Gln3, a transcription activator of catabolic genes whose products generate the nitrogenous precursors for protein synthesis, is not subject to leucine-dependent TorC1 activation. This led us to conclude that excess nitrogen-dependent down-regulation of Gln3 occurs via a second mechanism that is independent of leucine-dependent TorC1 activation. A major site of Gln3 and Gat1 (another GATA-binding transcription activator) control occurs at their access to the nucleus. In excess nitrogen, Gln3 and Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner. They become nuclear and activate transcription when nitrogen becomes limiting. Long-term nitrogen starvation and treatment of cells with the glutamine synthetase inhibitor methionine sulfoximine (Msx) also elicit nuclear Gln3 localization. The sensitivity of Gln3 localization to glutamine and inhibition of glutamine synthesis prompted us to investigate the effects of a glutamine tRNA mutation (sup70-65) on nitrogen-responsive control of Gln3 and Gat1. We found that nuclear Gln3 localization elicited by short- and long-term nitrogen starvation; growth in a poor, derepressive medium; Msx or rapamycin treatment; or ure2Δ mutation is abolished in a sup70-65 mutant. However, nuclear Gat1 localization, which also exhibits a glutamine tRNACUG requirement for its response to short-term nitrogen starvation or growth in proline medium or a ure2Δ mutation, does not require tRNACUG for its response to rapamycin. Also, in contrast with Gln3, Gat1 localization does not respond to long-term nitrogen starvation. These observations demonstrate the existence of a specific nitrogen-responsive component participating in the control of Gln3 and Gat1 localization and their downstream production of nitrogenous precursors. This component is highly sensitive to the function of the rare glutamine tRNACUG, which cannot be replaced by the predominant glutamine tRNACAA. Our observations also demonstrate distinct mechanistic differences between the responses of Gln3 and Gat1 to rapamycin inhibition of TorC1 and nitrogen starvation.

Nitrogen-responsive control of Gln3 localization is implemented through TorC1-dependent (rapamycin-responsive) and TorC1-independent (nitrogen catabolite repression-sensitive and methionine sulfoximine (Msx)-responsive) regulatory pathways. We previously demonstrated amino acid substitutions in a putative Gln3 α-helix(656-666), which are required for a two-hybrid Gln3-Tor1 interaction, also abolished rapamycin responsiveness of Gln3 localization and partially abrogated cytoplasmic Gln3 sequestration in cells cultured under nitrogen-repressive conditions. Here, we demonstrate these three characteristics are not inextricably linked together. A second distinct Gln3 region (Gln3(510-589)) is specifically required for rapamycin responsiveness of Gln3 localization, but not for cytoplasmic Gln3 sequestration under repressive growth conditions or relocation to the nucleus following Msx addition. Aspartate or alanine substitution mutations throughout this region uniformly abolish rapamycin responsiveness. Contained within this region is a sequence with a predicted propensity to form an α-helix(583-591), one side of which consists of three hydrophobic amino acids flanked by serine residues. Substitution of aspartate for even one of these serines abolishes rapamycin responsiveness and increases rapamycin resistance without affecting either of the other two Gln3 localization responses. In contrast, alanine substitutions decrease rapamycin resistance. Together, these data suggest that targets in the C-terminal portion of Gln3 required for the Gln3-Tor1 interaction, cytoplasmic Gln3 sequestration, and Gln3 responsiveness to Msx addition and growth in poor nitrogen sources are distinct from those needed for rapamycin responsiveness.

The GATA family transcription activator, Gln3 responds to the nitrogen requirements and environmental resources of the cell. When rapidly utilized, "good" nitrogen sources, e.g., glutamine, are plentiful, Gln3 is completely sequestered in the cytoplasm, and the transcription it mediates is minimal. In contrast, during nitrogen-limiting conditions, Gln3 quickly relocates to the nucleus and activates transcription of genes required to scavenge alternative, "poor" nitrogen sources, e.g., proline. This physiological response has been designated nitrogen catabolite repression (NCR). Because rapamycin treatment also elicits nuclear Gln3 localization, TorC1 has been thought to be responsible for NCR-sensitive Gln3 regulation. However, accumulating evidence now suggests that GATA factor regulation may occur by two separate pathways, one TorC1-dependent and the other NCR-sensitive. Therefore, the present experiments were initiated to identify Gln3 amino acid substitutions capable of dissecting the individual contributions of these pathways to overall Gln3 regulation. The rationale was that different regulatory pathways might be expected to operate through distinct Gln3 sensor residues. We found that C-terminal truncations or amino acid substitutions in a 17-amino acid Gln3 peptide with a predicted propensity to fold into an α-helix partially abolished the ability of the cell to sequester Gln3 in the cytoplasm of glutamine-grown cells and eliminated the rapamycin response of Gln3 localization, but did not adversely affect its response to limiting nitrogen. However, overall wild type control of intracellular Gln3 localization requires the contributions of both individual regulatory systems. We also found that Gln3 possesses at least one Tor1-interacting site in addition to the one previously reported.

Background and objectivesProgesterone is essential to maintain a healthy pregnancy. Guidance from the Royal College of Obstetricians and Gynaecologists and a Cochrane review called for a definitive trial to test whether or not progesterone therapy in the first trimester could reduce the risk of miscarriage in women with a history of unexplained recurrent miscarriage (RM). The PROMISE trial was conducted to answer this question. A concurrent cost-effectiveness analysis was conducted.Design and settingA randomised, double-blind, placebo-controlled, international multicentre study, with economic evaluation, conducted in hospital settings across the UK (36 sites) and in the Netherlands (nine sites).Participants and interventionsWomen with unexplained RM (three or more first-trimester losses), aged between 18 and 39 years at randomisation, conceiving naturally and giving informed consent, received either micronised progesterone (Utrogestan®, Besins Healthcare) at a dose of 400 mg (two vaginal capsules of 200 mg) or placebo vaginal capsules twice daily, administered vaginally from soon after a positive urinary pregnancy test (and no later than 6 weeks of gestation) until 12 completed weeks of gestation (or earlier if the pregnancy ended before 12 weeks).Main outcome measuresLive birth beyond 24 completed weeks of gestation (primary outcome), clinical pregnancy at 6–8 weeks, ongoing pregnancy at 12 weeks, miscarriage, gestation at delivery, neonatal survival at 28 days of life, congenital abnormalities and resource use.MethodsParticipants were randomised after confirmation of pregnancy. Randomisation was performed online via a secure internet facility. Data were collected on four occasions of outcome assessment after randomisation, up to 28 days after birth.ResultsA total of 1568 participants were screened for eligibility. Of the 836 women randomised between 2010 and 2013, 404 received progesterone and 432 received placebo. The baseline data (age, body mass index, maternal ethnicity, smoking status and parity) of the participants were comparable in the two arms of the trial. The follow-up rate to primary outcome was 826 out of 836 (98.8%). The live birth rate in the progesterone group was 65.8% (262/398) and in the placebo group it was 63.3% (271/428), giving a relative risk of 1.04 (95% confidence interval 0.94 to 1.15;p = 0.45). There was no evidence of a significant difference between the groups for any of the secondary outcomes. Economic analysis suggested a favourable incremental cost-effectiveness ratio for decision-making but wide confidence intervals indicated a high level of uncertainty in the health benefits. Additional sensitivity analysis suggested the probability that progesterone would fall within the National Institute for Health and Care Excellence’s threshold of £20,000–30,000 per quality-adjusted life-year as between 0.7145 and 0.7341.ConclusionsThere is no evidence that first-trimester progesterone therapy improves outcomes in women with a history of unexplained RM.LimitationsThis study did not explore the effect of treatment with other progesterone preparations or treatment during the luteal phase of the menstrual cycle.Future workFuture research could explore the efficacy of progesterone supplementation administered during the luteal phase of the menstrual cycle in women attempting natural conception despite a history of RM.Trial registrationCurrent Controlled Trials ISRCTN92644181; EudraCT 2009-011208-42; Research Ethics Committee 09/H1208/44.FundingThis project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full inHealth Technology Assessment; Vol. 20, No. 41. See the NIHR Journals Library website for further project information.

A remarkable characteristic of nutritional homeostatic mechanisms is the breadth of metabolite concentrations to which they respond, and the resolution of those responses; adequate but rarely excessive. Two general ways of achieving such exquisite control are known: stoichiometric mechanisms where increasing metabolite concentrations elicit proportionally increasing responses, and the actions of multiple independent metabolic signals that cumulatively generate appropriately measured responses. Intracellular localization of the nitrogen-responsive transcription activator, Gln3, responds to four distinct nitrogen environments: nitrogen limitation or short-term starvation, i.e., nitrogen catabolite repression (NCR), long-term starvation, glutamine starvation, and rapamycin inhibition of mTorC1. We have previously identified unique sites in Gln3 required for rapamycin-responsiveness, and Gln3-mTor1 interaction. Alteration of the latter results in loss of about 50% of cytoplasmic Gln3 sequestration. However, except for the Ure2-binding domain, no evidence exists for a Gln3 site responsible for the remaining cytoplasmic Gln3-Myc13 sequestration in nitrogen excess. Here, we identify a serine/threonine-rich (Gln3477–493) region required for effective cytoplasmic Gln3-Myc13 sequestration in excess nitrogen. Substitutions of alanine but not aspartate for serines in this peptide partially abolish cytoplasmic Gln3 sequestration. Importantly, these alterations have no effect on the responses of Gln3-Myc13 to rapamycin, methionine sulfoximine, or limiting nitrogen. However, cytoplasmic Gln3-Myc13 sequestration is additively, and almost completely, abolished when mutations in the Gln3-Tor1 interaction site are combined with those in Gln3477–493 cytoplasmic sequestration site. These findings clearly demonstrate that multiple individual regulatory pathways cumulatively control cytoplasmic Gln3 sequestration.

Gln3, a transcription activator mediating nitrogen-responsive gene expression in Saccharomyces cerevisiae, is sequestered in the cytoplasm, thereby minimizing nitrogen catabolite repression (NCR)-sensitive transcription when cells are grown in nitrogen-rich environments. In the face of adverse nitrogen supplies, Gln3 relocates to the nucleus and activates transcription of the NCR-sensitive regulon whose products transport and degrade a variety of poorly used nitrogen sources, thus expanding the cell’s nitrogen-acquisition capability. Rapamycin also elicits nuclear Gln3 localization, implicating Target-of-rapamycin Complex 1 (TorC1) in nitrogen-responsive Gln3 regulation. However, we long ago established that TorC1 was not the sole regulatory system through which nitrogen-responsive regulation is achieved. Here we demonstrate two different ways in which intracellular Gln3 localization is regulated. Nuclear Gln3 entry is regulated by the cell’s overall nitrogen supply, i.e., by NCR, as long accepted. However, once within the nucleus, Gln3 can follow one of two courses depending on the glutamine levels themselves or a metabolite directly related to glutamine. When glutamine levels are high, e.g., glutamine or ammonia as the sole nitrogen source or addition of glutamine analogues, Gln3 can exit from the nucleus without binding to DNA. In contrast, when glutamine levels are lowered, e.g., adding additional nitrogen sources to glutamine-grown cells or providing repressive nonglutamine nitrogen sources, Gln3 export does not occur in the absence of DNA binding. We also demonstrate that Gln3 residues 64–73 are required for nuclear Gln3 export.

Vam6, Gtr1/2, and Ego1/3 are required for leucine-dependent TorC1 kinase activation which is central to nitrogen-responsive regulation. However, Gln3, a nitrogen-responsive transcription activator, does not respond to leucine-dependent TorC1 activation. In nitrogen excess, Gln3 is cytoplasmic and Gln3-mediated transcription minimal, whereas in nitrogen limitation, starvation, or following rapamycin treatment, Gln3 is nuclear and transcription greatly increased. Increasing evidence demonstrates nitrogen-responsive intracellular Gln3 localization is subject to multiple modes of regulation. To ascertain whether the Vam6, Gtr-Ego complexes participate in the regulation of Gln3, we determined the requirements of the above proteins for nuclear localization and cytoplasmic sequestration of Gln3 in response to nitrogen excess, starvation or limitation. We show that Gln3 is sequestered in the cytoplasm of vam6Δ, gtr1Δ, gtr2Δ, ego1Δ and ego3Δ either long-term in logarithmically glutamine-grown cells or short-term a...

A leucine-dependent pathway activates TorC1 kinase and its downstream stimulation of protein synthesis. However, Gln3 and Gat1, nitrogen-responsive transcription activators, don't respond to leucine-dependent TorC1 activation. This led us to conclude that excess nitrogen-dependent down regulation of Gln3 occurs via a second mechanism. A major site of Gln3/Gat1 control occurs at their access to the nucleus. In excess nitrogen, Gln3/Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner. They become nuclear when nitrogen or glutamine is limiting. The sensitivity of Gln3 localization to glutamine and inhibition of glutamine synthesis by methionine sulfoximine (Msx) prompted us to investigate the effects of a glutamine tRNA mutation (sup70-65) on Gln3/Gat1 regulation. Contrary to prediction, we show nuclear Gln3 localization - elicited by short- and long-term nitrogen starvation, growth in proline, during Msx or rapamycin treatment or in a ure2Δ - is abolished by alteration of glutamine tRNACUG. In ...

Gln3 and Gat1/Nil1 are GATA-family transcription factors responsible for transcription of nitrogen-catabolic genes in Saccharomyces cerevisiae. Intracellular Gln3 localization and Gln3-dependent transcription respond in parallel to the nutritional environment and inhibitors of Tor1/2 (rapamycin) and glutamine synthetase (L-methionine sulfoximine, MSX). However, detectable Gln3 phosphorylation, though influenced by nutrients and inhibitors, correlates neither with Gln3 localization nor nitrogen catabolite repression-sensitive transcription in a consistent way. To establish relationships between Gln3 and Gat1 regulation, we performed experiments parallel to those we previously reported for Gln3. Gat1 and Gln3 localization are similar during steady-state growth, being cytoplasmic and nuclear with good and poor nitrogen sources, respectively. Localization correlates with Gat1- and Gln3-mediated transcription. In contrast, three characteristics of Gat1 and Gln3 differ significantly: (i) the kinetics of their localization in response to nutritional transitions and rapamycin-treatment; (ii) their opposite responses to MSX-treatment, i.e. that cytoplasmic Gln3 becomes nuclear following MSX addition, whereas nuclear Gat1 becomes cytoplasmic; and (iii) their phosphorylation levels in the above situations. In instances where Gln3 phosphorylation can be straightforwardly demonstrated to change, Gat1 phosphorylation (in the same samples) appears invariant. The only exception was following carbon starvation, where Gat1, like Gln3, is hyperphosphorylated in a Snf1-dependent manner. However, neither carbon starvation nor MSX treatment elicits Snf1-independent Gat1 hyperphosphorylation, as observed for Gln3.

The Saccharomyces cerevisiae allantoate/ureidosuccinate permease gene (DAL5) is often used as a reporter in studies of the Tor1/2 protein kinases which are specifically inhibited by the clinically important immunosuppressant and anti-neoplastic drug, rapamycin. To date, only a single type of cis-acting element has been shown to be required for DAL5 expression, two copies of the GATAA-containing UASNTR element that mediates nitrogen catabolite repression-sensitive transcription. UASNTR is the binding site for the transcriptional activator, Gln3 whose intracellular localization responds to the nitrogen supply, accumulating in the nuclei of cells provided with poor nitrogen sources and in the cytoplasm when excess nitrogen is available. Recent data raised the possibility that DAL5 might also be regulated by the retrograde system responsible for control of early TCA cycle gene expression, prompting us to investigate the structure of the DAL5 promoter in more detail. Here, we show that clearly one (UASB), and possibly two (UASA), additional cis-acting elements are required for full DAL5 expression. One of these elements (UASB) is in a region that is heavily protected from DNaseI digestion and functions in a highly synergistic manner with the two UASNTR elements. Cis-acting elements UASNTR–UASA and UASNTR–UASB are situated on the same face of the DNA two and one turn apart, respectively. We also found that decreased DAL5 expression in glutamate-grown cells, a characteristic shared with retrograde regulation, likely derives from decreased nuclear Gln3 levels that occur under these growth conditions rather than direct retrograde system control.

Ure2, the protein that negatively regulates GATA factor (Gln3, Gat1)-mediated transcription in Saccharomyces cerevisiae, possesses prion-like characteristics. Identification of metabolic and environmental factors that influence prion formation as well as any activities that prions or prion precursors may possess are important to understanding them and developing treatment strategies for the diseases in which they participate. Ure2 exhibits primary sequence and three-dimensional homologies to known glutathione S-transferases. However, multiple attempts over nearly 2 decades to demonstrate Ure2-mediated S-transferase activity have been unsuccessful, leading to the possibility that Ure2 may well not participate in glutathionation reactions. Here we show that Ure2 is required for detoxification of glutathione S-transferase substrates and cellular oxidants. ure2 Delta mutants are hypersensitive to cadmium and nickel ions and hydrogen peroxide. They are only slightly hypersensitive to diamide, which is nitrogen source-dependent, and minimally if at all hypersensitive to 1-chloro-2,4-dinitrobenzene, the most commonly used substrate for glutathione S-transferase enzyme assays. Therefore, Ure2 shares not only structural homology with various glutathione S-transferases, but ure2 mutations possess the same phenotypes as mutations in known S. cerevisiae and Schizosaccharomyces pombe glutathione S-transferase genes. These findings are consistent with Ure2 serving as a glutathione S-transferase in S. cerevisiae.

The Gtr1/2, Ego1/3 complexes and Vam6 have been reported to be required for TorC1 kinase activation. In turn, activated TorC1 is accepted to be required for cytoplasmic Gln3 sequestration based on ...

Nitrogen catabolite repression (NCR)-sensitive transcription is activatedby Gln3 and Gat1. Innitrogen excess, Gln3 and Gat1 are cytoplasmic, and transcription is minimal. In poor nitrogen, Gln3 and Gat1 become nuclear and activate transcription. A long standing paradox has surrounded Gat1 production. Gat1 was first reported as an NCR-regulated activity mediating NCR-sensitive transcription in gln3 deletion strains. Upon cloning, GAT1 transcription was, as predicted, NCR-sensitive and Gln3- and Gat1-activated. In contrast, Western blots of Gat1-Myc13 exhibited two constitutively produced species. Investigating this paradox, wedemonstrate that wild type Gat1 isoforms (IsoA and IsoB) are initiated at Gat1 methionines 40, 95, and/or 102, but not at methionine 1. Their low level production is the same in rich and poor nitrogen conditions. When the Myc13 tag is placed after Gat1 Ser-233, four N-terminal Gat1 isoforms (IsoC-F) are also initiated at methionines 40, 95, and/or 102. However, their production is highly NCR-sensitive, being greater in proline than glutamine medium. Surprisingly, all Gat1 isoforms produced in sufficient quantities to be confidently analyzed (IsoA, IsoC, and IsoD) require Gln3 and UASGATA promoter elements, both requirements typical of NCR-sensitive transcription. These data demonstrate that regulated Gat1 production is more complex than previously recognized, with wild type versus truncated Gat1 proteins failing to be regulated in parallel. This is the first reported instance of Gln3 UAS GATA-dependent protein production failing to derepress in nitrogen poor conditions. A Gat1-lacZ ORF swap experiment indicated sequence(s) responsible for the nonparallel production are downstream of Gat1 leucine 61. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc., SCOPUS: ar.j, info:eu-repo/semantics/published

Nitrogen-catabolic gene expression in Saccharomyces cerevisiae is regulated by the action of four GATA family transcription factors: Gln3p and Gat1p/Nil1p are transcriptional activators, and Dal80 and Deh1p/Gzf3p are repressors. In addition to the GATA sequences situated upstream of all nitrogen catabolite repression-sensitive genes that encode enzyme and transport proteins, the promoters of the GAT1 , DAL80 , and DEH1 genes all contain multiple GATA sequences as well. These GATA sequences are the binding sites of the GATA family transcription factors and are hypothesized to mediate their autogenous and cross regulation. Here we show, using DAL80 fused to the carbon-regulated GAL1 , 10 or copper-regulated CUP1 promoter, that GAT1 expression is inversely regulated by the level of DAL80 expression, i.e., as DAL80 expression increases, GAT1 expression decreases. The amount of DAL80 expression also dictates the level at which DAL3 , a gene activated almost exclusively by Gln3p, is transcribed. Gat1p was found to partially substitute for Gln3p in transcription. These data support the contention that regulation of GATA-factor gene expression is tightly and dynamically coupled. Finally, we suggest that the complicated regulatory circuit in which the GATA family transcription factors participate is probably most beneficial as cells make the transition from excess to limited nitrogen availability.

Dal82p binds to the UIS ALL sites of allophanate-induced genes of the allantoin-degradative pathway and functions synergistically with the GATA family Gln3p and Gat1p transcriptional activators that are responsible for nitrogen catabolite repression-sensitive gene expression. CAR2 , which encodes the arginine-degradative enzyme ornithine transaminase, is not nitrogen catabolite repression sensitive, but its expression can be modestly induced by the allantoin pathway inducer. The dominant activators of CAR2 transcription have been thought to be the ArgR and Mcm1 factors, which mediate arginine-dependent induction. These observations prompted us to investigate the structure of the CAR2 promoter with the objectives of determining whether other transcription factors were required for CAR2 expression and, if so, of ascertaining their relative contributions to CAR2 ’s expression and control. We show that Rap1p binds upstream of CAR2 and plays a central role in its induced expression irrespective of whether the inducer is arginine or the allantoin pathway inducer analogue oxalurate (OXLU). Our data also explain the early report that ornithine transaminase production is induced when cells are grown with urea. OXLU induction derives from the Dal82p binding site, which is immediately downstream of the Rap1p site, and Dal82p functions synergistically with Rap1p. This synergism is unlike all other known instances of Dal82p synergism, namely, that with the GATA family transcription activators Gln3p and Gat1p, which occurs only in the presence of an inducer. The observations reported suggest that CAR2 gene expression results from strong constitutive transcriptional activation mediated by Rap1p and Dal82p being balanced by the down regulation of an equally strong transcriptional repressor, Ume6p. This balance is then tipped in the direction of expression by the presence of the inducer. The formal structure of the CAR2 promoter and its operation closely follow the model proposed for CAR1 .

Nitrogen catabolic gene expression in Saccharomyces cerevisiae has been reported to be regulated by three GATA family proteins, the positive regulators Gln3p and Gat1p/Nil1p and the negative regulator Dal80p/Uga43p. We show here that a fourth member of the yeast GATA family, the Dal80p homolog Deh1p, also negatively regulates expression of some, but not all, nitrogen catabolic genes, i.e., GAP1, DAL80, and UGA4 expression increases in a deh1 delta mutant. Consistent with Deh1p regulation of these genes is the observation that Deh1p forms specific DNA-protein complexes with GATAA-containing UGA4 and GAP1 promoter fragments in electrophoretic mobility shift assays. Deh1p function is demonstrable, however, only when a repressive nitrogen source such as glutamine is present; deh1 delta mutants exhibit no detectable phenotype with a poor nitrogen source such as proline. Our experiments also demonstrate that GATA factor gene expression is highly regulated by the GATA factors themselves in an interdependent manner. DAL80 expression is Gln3p and Gat1p dependent and Dal80p regulated. Moreover, Gln3p and Dal80p bind to DAL80 promoter fragments. In turn, GAT1 expression is Gln3p dependent and Dal80p regulated but is not autogenously regulated like DAL80. DEH1 expression is largely Gln3p independent, modestly Gat1p dependent, and most highly regulated by Dal80p. Paradoxically, the high-level DEH1 expression observed in a dal80::hisG disruption mutant is highly sensitive to nitrogen catabolite repression.

When readily used nitrogen sources are available, the expression of genes encoding proteins needed to transport and metabolize poorly used nitrogen sources is repressed to low levels; this physiological response has been designated nitrogen catabolite repression (NCR). The cis-acting upstream activation sequence (UAS) element UAS(NTR) mediates Gln3p-dependent, NCR-sensitive transcription and consists of two separated dodecanucleotides, each containing the core sequence GATAA. Gln3p, produced in Escherichia coli and hence free of all other yeast proteins, specifically binds to wild-type UAS(NTR) sequences and DNA fragments derived from a variety of NCR-sensitive promoters (GDH2, CAR11 DAL3, PUT1, UGA4, and GLN1). A LexA-Gln3 fusion protein supported transcriptional activation when bound to one or more LexAp binding sites upstream of a minimal CYC1-derived promoter devoid of UAS elements. LexAp-Gln3p activation of transcription was largely independent of the nitrogen source used for growth. These data argue that Gln3p is capable of direct UAS(NTR) binding and participates in transcriptional activation of NCR-sensitive genes.

UASNTR, the UAS responsible for nitrogen catabolite repression-sensitive transcriptional activation of many nitrogen catabolic genes in Saccharomyces cerevisiae, has been previously thought to operate only as a pair of closely related dodecanucleotide sites each containing the sequence GATAA at its core. Here we show that a single UASNTR site is also able to combine with another unrelated cis-acting element to mediate transcription as well. In one instance the unrelated cis-acting element was TTTGTTTAC situated upstream of GLN1, while in another the cis-acting element was the one previously shown to bind the PUT3 protein. When a UASNTR site functions in combination with an unrelated site, the regulatory responses observed are a hybrid consisting of characteristics derived from both the UASNTR site and the unrelated site as well. These observations resolve several significant inconsistencies that have plagued studies focused on elucidation of the mechanisms involved in the global regulation of nitrogen catabolism.

Gene expression during lytic development of bacteriophage Mu occurs in three phases: early, middle, and late. Transcription from the middle promoter, P(m), requires the phage-encoded activator protein Mor and the bacterial RNA polymerase. The middle promoter has a -10 hexamer, but no -35 hexamer. Instead P(m) has a hyphenated inverted repeat that serves as the Mor binding site overlapping the position of the missing -35 element. Mor binds to this site as a dimer and activates transcription by recruiting RNA polymerase. The crystal structure of the His-Mor dimer revealed three structural elements: an N-terminal dimerization domain, a C-terminal helix-turn-helix DNA-binding domain, and a β-strand linker between the two domains. We predicted that the highly conserved residues in and flanking the β-strand would be essential for the conformational flexibility and DNA minor groove binding by Mor. To test this hypothesis, we carried out single codon-specific mutagenesis with degenerate oligonucleotides. The amino acid substitutions were identified by DNA sequencing. The mutant proteins were characterized for their overexpression, solubility, DNA binding, and transcription activation. This analysis revealed that the Gly-Gly motif formed by Gly-65 and Gly-66 and the β-strand side chain of Tyr-70 are crucial for DNA binding by His-tagged Mor. Mutant proteins with substitutions at Gly-74 retained partial activity. Treatment with the minor groove- and GC-specific chemical chromomycin A(3) demonstrated that chromomycin prevented His-Mor binding but could not disrupt a pre-formed His-Mor·DNA complex, consistent with the prediction that Mor interacts with the minor groove of the GC-rich spacer in the Mor binding site.

We demonstrate that expression of the UGA1, CAN1, GAP1, PUT1, PUT2, PUT4, and DAL4 genes is sensitive to nitrogen catabolite repression. The expression of all these genes, with the exception of UGA1 and PUT2, also required a functional GLN3 protein. In addition, GLN3 protein was required for expression of the DAL1, DAL2, DAL7, GDH1, and GDH2 genes. The UGA1, CAN1, GAP1, and DAL4 genes markedly increased their expression when the DAL80 locus, encoding a negative regulatory element, was disrupted. Expression of the GDH1, PUT1, PUT2, and PUT4 genes also responded to DAL80 disruption, but much more modestly. Expression of GLN1 and GDH2 exhibited parallel responses to the provision of asparagine and glutamine as nitrogen sources but did not follow the regulatory responses noted above for the nitrogen catabolic genes such as DAL5. Steady-state mRNA levels of both genes did not significantly decrease when glutamine was provided as nitrogen source but were lowered by the provision of asparagine. They also did not respond to disruption of DAL80.

The prevalence of congenital heart disease (CHD) is not known in our country. The aim of present study was to find out the prevalence of CHD in school children of eastern Uttar Pradesh.A team consisting of a cardiologist, physicians and junior residents visited schools in the area. All the children were examined for presence of cardiac murmur or history of heart disease or any intervention. Those with murmurs or previous history of heart disease were called to the Medical College Hospital for evaluation by ECG, chest X-ray and echocardiography for confirmation of the lesion.Out of 118,212 children examined, 142 were found to have CHD. The prevalence was 1.3 per 1000 children and the commonest lesions were ventricular and atrial septal defects, aortic stenosis with or without regurgitation, and pulmonary stenosis.CHD prevalence is 1.3 per 1000 school children that is nearly two and a half times more than that of RHD. Knowing it is important for development of facilities for CHD care in our setup.

Rheumatic heart disease is a major health problem in our country. There is evidence from South India that its prevalence is declining. This study attempts to confirm whether this is so in North India as well.A total of 118,212 (68,357 males, 49,855 females) schoolchildren in the age group of 4-18 years were examined for the presence of heart disease. Evaluation, including echocardiography, confirmed that of a total of 389 suspected to have heart disease, 61 had rheumatic heart disease. Thus, the prevalence of rheumatic heart disease was found to be approximately 0.5 per 1000 children.In a fairly large school survey conducted by us, the prevalence of rheumatic heart disease turned out to be approximately 0.5 per 1000 children. This is the lowest figure reported from our country so far and confirms the decline of this disease in our country.

Gamma-aminobutyrate (GABA) accumulation in growing cultures of Saccharomyces cerevisiae was shown to occur by means of an active transport system that is inhibited by proton ionophores, azide, fluoride and arsenate ions. Transport occurred maximally at pH 5.0 and exhibited apparent Km values of 12 microM and 0.1 mM. Accumulated GABA did not efflux upon treatment with proton ionophores and exchanged with extracellular material only very slowly. However, release was complete upon treatment with nystatin. These observations raise the possibility that a major portion of intracellular GABA is sequestered in the vacuole. The response of GABA uptake to growth on various nitrogen sources suggested that uptake may be subject to several types of regulation.

We show that mutation at the GLN3 locus results in decreased steady-state levels of DAL7, DUR1,2, CAR1, and URA3 mRNAs derived from cultures grown in the presence of inducer. Basal levels of these RNA species, however, were not significantly affected by a gln3 mutation. The GLN3 product appears to affect gene expression in two ways. The pleiotropic requirement of GLN3 for induced gene expression probably derives from the need of the GLN3 product for inducer uptake into the cell and its loss in gln3 mutants. We also demonstrate that transcriptional activation, mediated by the DAL5 and DAL7 upstream activation sequences, requires a functional GLN3 gene product. This observation identified transcriptional activation as the most likely point of GLN3 participation in the expression of allantoin system genes.

Events directly regulating Gln3 intracellular localization and nitrogen catabolite repression (NCR)-sensitive transcription in Saccharomyces cerevisiae are interconnected with many cellular processes that influence the utilization of environmental metabolites. Among them are intracellular trafficking of the permeases that transport nitrogenous compounds and their control by the Tor1,2 signal transduction pathway. Npr1 is a kinase that phosphorylates and thereby stabilizes NCR-sensitive permeases, e.g. Gap1 and Mep2. It is also a phosphoprotein for which phosphorylation and kinase activity are regulated by Tor1,2 via Tap42 and Sit4. Npr1 has been reported to negatively regulate nuclear localization of Gln3 in SD (ammonia)-grown cells. Thus we sought to distinguish whether Npr1: (i) functions directly as a component of NCR control; or (ii) influences Gln3 localization indirectly, possibly as a consequence of participating in protein trafficking. If Npr1 functions directly, then the ability of all good nitrogen sources to restrict Gln3 to the cytoplasm should be lost in an npr1Delta just as occurs when URE2 (encoding this well studied negative Gln3 regulator) is deleted. We show that nuclear localization of Gln3-Myc(13) in an npr1Delta occurred only with ammonia as the nitrogen source. Other good nitrogen sources, e.g. glutamine, serine, or asparagine, restricted Gln3-Myc(13) to the cytoplasm of both wild type and npr1Delta cells. In other words, the npr1Delta did not possess the uniform phenotype for all repressive nitrogen sources characteristic of ure2Delta. This suggests that the connection between Gln3 localization and Npr1 is indirect, arising from the influence of Npr1 on the ability of cells to utilize ammonia as a repressive nitrogen source.

Gln3 and Gat1/Nil1 are GATA-family transcription factors responsible for transcription of nitrogen catabolic genes in S. cerevisiae. Intracellular Gln3 localization and Gln3-dependent transcription respond in parallel to the nutritional environment and inhibitors of Tor1/2 (rapamycin) and glutamine synthetase (methionine sulfoximine, MSX). However, detectable Gln3 phosphorylation, though influenced by nutrients and inhibitors, does not correlate with Gln3 localization or NCR-sensitive transcription in a consistent way. Little such data are available for Gat1. To eliminate this gap and establish relationships between Gln3 and Gat1 regulation, we performed experiments parallel to those we previously reported for Gln3. Gat1 and Gln3 localization are similar during steady state growth, being cytoplasmic and nuclear with good and poor nitrogen sources, respectively. Localization correlates with Gat1- and Gln3-mediated transcription. In contrast, three characteristics of Gat1 and Gln3 differ significantly: (i) the kinetics of their localization in response to nutritional transitions and rapamycin-treatment, (ii) their opposite responses to MSX-treatment, i.e., cytoplasmic Gln3 becomes nuclear following MSX addition, while nuclear Gat1 becomes cytoplasmic, and (iii) their phosphorylation levels in the above situations. In instances where Gln3 phosphorylation can be straightforwardly demonstrated to change, Gat1 phosphorylation (in the same samples) appears invariant. The only exception was following carbon starvation where Gat1, like Gln3, is hyperphosphorylated in a Snf1-dependent manner. However, neither carbon-starvation, nor MSX-treatment elicits Snf1-independent Gat1 hyperphosphorylation as observed for Gln3.

Tor proteins are global regulators situated at the top of a signal transduction pathway conserved from yeast to humans. Specific inhibition of the two Saccharomyces cerevisiae Tor proteins by rapamycin alters many cellular processes and the expression of hundreds of genes. Among the regulated genes are those whose expression is activated by the GATA family transcription activator, Gln3. The extent of Gln3 phosphorylation has been thought to determine its intracellular localization, with phosphorylated and dephosphorylated forms accumulating in the cytoplasm and nucleus, respectively. Data presented here demonstrate that rapamycin and the glutamine synthetase inhibitor, methionine sulfoximine (MSX), although eliciting the same outcomes with respect to Gln3-Myc13 nuclear accumulation and nitrogen catabolite repression-sensitive transcription, generate diametrically opposite effects on Gln3-Myc13 phosphorylation. MSX increases Gln3-Myc13 phosphorylation and rapamycin decreases it. Gln3-Myc13 phosphorylation levels are regulated by at least three mechanisms as follows: (i) depends on Snf1 kinase as observed during carbon starvation, (ii) is Snf1-independent as observed during both carbon starvation and MSX treatment, and (iii) is rapamycin-induced dephosphorylation. MSX and rapamycin act additively on Gln3-Myc13 phosphorylation, but MSX clearly predominates. These results suggest that MSX- and rapamycin-inhibited proteins are more likely to function in separate regulatory pathways than they are to function tandemly in a single pathway as thought previously. Furthermore, as we and others have detected thus far, Gln3 phosphorylation/dephosphorylation is not a demonstrably required step in achieving Gln3 nuclear localization and nitrogen catabolite repression-sensitive transcription in response to MSX or rapamycin treatment.

The S. cerevisiae Ure2 protein is a prion precursor able to form large homopolymers with the characteristics of amyloid particles, a function largely restricted to its 90 N-terminal amino acids. The remaining C-terminal domain of Ure2 plays two important roles in cellular metabolism. First, it regulates nitrogen catabolic gene expression by forming a complex with the GATA transcription factor Gln3. This complex formation correlates with Gln3 being sequestered in the cytoplasm under conditions of excess nitrogen, where Gln3/Gat1-mediated transcription is minimal. Second, Ure2, which possesses structural homology with glutathione S-transferases and binds to xenobiotics and glutathione, has been recently shown to be required for Cd(II) and hydrogen peroxide detoxification. Present experiments demonstrate that Ure2 possesses a far broader protection specificity, being required to avoid the toxic effects of As(III), As(V), Cr(III), Cr(VI), Se(IV), as well as Cd(II) and Ni(II), and to varying lesser degrees Co(II), Cu(II), Fe(II), Ag(I), Hg(II), cumene and t-butyl hydroperoxides. In contrast, deletion of URE2 greatly enhances a cell's ability to withstand toxic concentrations of Zn(II) and Mo(VI). In the case of Cd(II), Ure2 does not function to decrease intracellular Cd(II) levels or influence glutathione availability for glutathionation. In fact, ure2 hypersensitivity to Cd(II) remains the same, even when glutathione is used as sole source of nitrogen for cell growth. These data suggest that Ure2 possesses a central role in metal ion detoxification, a role not demonstrably shared by either of the two known S. cerevisiae glutathione S-transferases, Gtt1 and Gtt2, or the two glutaredoxins, Grx1 and Grx2, that also possess glutathione S-transferase activity. Copyright © 2005 John Wiley & Sons, Ltd.

The Tor1/2p signal transduction pathway regulates nitrogen catabolite repression (NCR)-sensitive (GAP1, GAT1, DAL5) and retrograde (CIT2, DLD3, IDH1/2) gene expression by controlling intracellular localization of the transcription activators, Gln3p and Gat1p, and Rtg1p and Rtg3p, respectively. The accepted pathway for this regulation is NH(3) or excess nitrogen dash, vertical Mks1p dash, vertical Ure2p dash, vertical Gln3p --DAL5, and rapamycin or limiting nitrogen dash, vertical Torp --Tap42 dash, vertical Mks1p --Rtg1/3p --CIT2, respectively. In current models, Mks1p positively regulates both Gln3p (and DAL5 expression) and Rtg1/3p (and CIT2 expression). Here, in contrast, we show the following. (i) Mks1p is a strong negative regulator of CIT2 expression and does not effect NCR-sensitive expression of DAL5 or GAP1. (ii) Retrograde carbon and NCR-sensitive nitrogen metabolism are not linked by the quality of the nitrogen source, i.e. its ability to elicit NCR, but by the product of its catabolism, i.e. glutamate or ammonia. (iii) In some instances, we can dissociate rapamycin-induced CIT2 expression from Mks1p function, i.e. rapamycin does not suppress Mks1p-mediated down-regulation of CIT2 expression. These findings suggest that currently accepted models of Tor1/2p signal transduction pathway regulation require revision.

Gln3p is one of two well characterized GATA family transcriptional activation factors whose function is regulated by the nitrogen supply of the cell. When nitrogen is limiting, Gln3p and Gat1p are concentrated in the nucleus where they bind GATA sequences upstream of nitrogen catabolite repression (NCR)-sensitive genes and activate their transcription. Conversely, in excess nitrogen, these GATA sequences are unoccupied by Gln3p and Gat1p because these transcription activators are excluded from the nucleus. Ure2p binds to Gln3p and Gat1p and is required for NCR-sensitive transcription to be repressed and for nuclear exclusion of these transcription factors. Here we show the following. (i) Gln3p residues 344–365 are required for nuclear localization. (ii) Replacing Ser-344, Ser-347, and Ser-355 with alanines has minimal effects on GFP-Gln3p localization. However, replacing Gln3p Ser-344, Ser-347, and Ser-355 with aspartates results in significant loss of its ability to be concentrated in the nucleus. (iii) N and C termini of the Gln3p region required for it to complex with Ure2p and be excluded from the nucleus are between residues 1–103 and 301–365, respectively. (iv) N and C termini of the Ure2p region required for it to interact with Gln3p are situated between residues 101–151 and 330–346, respectively. (v) Loss of Ure2p residues participating in either dimer or prion formation diminishes its ability to carry out NCR-sensitive regulation of Gln3p activity.

Saccharomyces cerevisiae selectively uses good nitrogen sources (glutamine) in preference to poor ones (proline) by repressing GATA factor-dependent transcription of the genes needed to transport and catabolize poor nitrogen sources, a physiological process designated nitrogen catabolite repression (NCR). We show that some NCR-sensitive genes (CAN1, DAL5, DUR1,2, and DUR3) produce two transcripts of slightly different sizes. Synthesis of the shorter transcript is NCR-sensitive and that of the longer transcript is not. The longer transcript also predominates in gln3Delta mutants irrespective of the nitrogen source provided. We demonstrate that the longer mRNA species arises through the use of an alternative transcription start site generated by Gln3p-binding sites (GATAAs) being able to act as surrogate TATA elements. The ability of GATAAs to serve as surrogate TATAs, i.e. when synthesis of the shorter, NCR-sensitive transcripts are inhibited, correlates with sequestration of enhanced green fluorescent protein (EGFP)-Gln3p in the cytoplasm in a way that is indistinguishable from that seen with EGFP-Ure2p. However, when the shorter, NCR-sensitive DAL5 transcript predominates, EGFP-Gln3p is nuclear. These data suggest that the mechanism underlying NCR involves the cytoplasmic association of Ure2p with Gln3p, an interaction that prevents Gln3p from reaching it is binding sites upstream of NCR-sensitive genes.

Allantoin pathway gene expression in Saccharomyces cerevisiae responds to two different environmental stimuli. The expression of these genes is induced in the presence of allantoin or its degradative metabolites and repressed when a good nitrogen source (e. g. asparagine or glutamine) is provided. Three types of cis-acting sites and trans-acting factors are required for allantoin pathway gene transcription as follows: (i) UAS(NTR) element associated with the transcriptional activators Gln3p and Gat1p, (ii) URS(GATA) element associated with the repressor Dal80p, and (iii) UIS(ALL) element associated with the Dal82 and Dal81 proteins required for inducer-dependent transcription. Most of the work leading to the above conclusions has employed inducer-independent allantoin pathway genes (e.g. DAL5 and DAL3). The purpose of this work is to extend our understanding of these elements and their roles to inducible allantoin pathway genes using the DAL7 (encoding malate synthase) as a model. We show that eight distinct cis-acting sites participate in the process as follows: a newly identified GC-rich element, two UAS(NTR), two UIS(ALL), and three URS(GATA) elements. The two GATA-containing UAS(NTR) elements are coincident with two of the three GATA sequences that make up the URS(GATA) elements. The remaining URS(GATA) GATA sequence, however, is not a UAS(NTR) element but appears to function only in repression. The data provide insights into how these cis- and trans-acting factors function together to accomplish the regulated expression of the DAL7 gene that is observed in vivo.

Saccharomyces cerevisiae cells selectively use nitrogen sources in their environment. Nitrogen catabolite repression (NCR) is the basis of this selectivity. Until recently NCR was thought to be accomplished exclusively through the negative regulation of Gln3p function by Ure2p. The demonstration that NCR-sensitive expression of multiple nitrogen-catabolic genes occurs in a gln3 delta ure2 delta dal80::hisG triple mutant indicated that the prevailing view of the nitrogen regulatory circuit was in need of revision; additional components clearly existed. Here we demonstrate that another positive regulator, designated Gat1p, participates in the transcription of NCR-sensitive genes and is able to weakly activate transcription when tethered upstream of a reporter gene devoid of upstream activation sequence elements. Expression of GAT1 is shown to be NCR sensitive, partially Gln3p dependent, and Dal80p regulated. In agreement with this pattern of regulation, we also demonstrate the existence of Gln3p and Dal80p binding sites upstream of GAT1.

NIT2, a positive-acting regulatory protein in Neurospora crassa, activates the expression of a series of unlinked structural genes that encode nitrogen catabolic enzymes. NIT2 binding sites in the promoter regions of nit3, alc and lao have at least two GATA sequence elements. We have examined the binding affinity of the NIT2 protein for the yeast DAL5 wild-type upstream activation sequence UASNTR, which contains two GATA elements, and for a series of mutated binding sites, each differing from the wild-type site by a single base. Substitution for individual nucleotides within 5′ or 3′ sequences that flank the GATA elements had only modest effects upon NIT2 binding. In contrast, nearly all substitutions within the GATA elements almost completely eliminated NIT2 binding, demonstrating the importance of the GATA sequence for NIT2 binding. Four high-affinity binding sites for the NIT2 protein were found within a central region of the nit-2 gene itself.