Reporter gene transactivation by human p53 is inhibited in thioredoxin reductase null yeast by a mechanism associated with thioredoxin oxidation and independent of changes in the redox state of glutathione.

Reporter gene transactivation by human p53 is compromised in S. cerevisiae lacking the TRR1 gene encoding thioredoxin reductase. The basis for p53 inhibition was investigated by measuring the redox state of thioredoxin and glutathione in wild-type and Deltatrr1 yeast. The Deltatrr1 mutation affected the redox state of both molecules. About 34% of thioredoxin was in the disulfide form in wild-type yeast and increased to 70% in Deltatrr1 yeast. About 18% of glutathione was in the GSSG form in wild-type yeast and increased to 32% in Deltatrr1 yeast. The Deltatrr1 mutation also resulted in a 2.9-fold increase in total glutathione per mg extract protein. Highcopy expression of the GLR1 gene encoding glutathione reductase in Deltatrr1 yeast restored the GSSG:GSH ratio to wild-type levels, but did not restore p53 activity. Also, p53 activity was shown to be unaffected by a Deltaglr1 mutation, even though the mutation was known to result in glutathione oxidation. In summary, the results show that, although glutathione becomes more oxidized in Deltatrr1 cells, glutathione oxidation is neither sufficient nor necessary for p53 inhibition. The results indicate that p53 activity has a specific requirement for an intact thioredoxin system, rather than a general dependence on the intracellular reducing environment.

A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools.

In Saccharomyces cerevisiae, MEC1 and RAD53 are essential for cell growth and checkpoint function. Their essential role in growth can be bypassed by deletion of a novel gene, SML1, which functions after several genes whose overexpression also suppresses mec1 inviability. In addition, sml1 affects various cellular processes analogous to overproducing the large subunit of ribonucleotide reductase, RNR1. These include effects on mitochondrial biogenesis, on the DNA damage response, and on cell growth. Consistent with these observations, the levels of dNTP pools in sml1 delta strains are increased compared to wild-type. This effect is not due to an increase in RNR transcription. Finally, both in vivo and in vitro experiments show that Sml1 binds to Rnr1. We propose that Sml1 inhibits dNTP synthesis posttranslationally by binding directly to Rnr1 and that Mec1 and Rad53 are required to relieve this inhibition.

A glutathione reductase mutant of yeast accumulates high levels of oxidized glutathione and requires thioredoxin for growth.

A glutathione reductase null mutant of Saccharomyces cerevisiae was isolated in a synthetic lethal genetic screen for mutations which confer a requirement for thioredoxin. Yeast mutants that lack glutathione reductase (glr1 delta) accumulate high levels of oxidized glutathione and have a twofold increase in total glutathione. The disulfide form of glutathione increases 200-fold and represents 63% of the total glutathione in a glr1 delta mutant compared with only 6% in wild type. High levels of oxidized glutathione are also observed in a trx1 delta, trx2 delta double mutant (22% of total), in a glr1 delta, trx1 delta double mutant (71% of total), and in a glr1 delta, trx2 delta double mutant (69% of total). Despite the exceptionally high ratio of oxidized/reduced glutathione, the glr1 delta mutant grows with a normal cell cycle. However, either one of the two thioredoxins is essential for growth. Cells lacking both thioredoxins and glutathione reductase are not viable under aerobic conditions and grow poorly anaerobically. In addition, the glr1 delta mutant shows increased sensitivity to the thiol oxidant diamide. The sensitivity to diamide was suppressed by deletion of the TRX2 gene. The genetic analysis of thioredoxin and glutathione reductase in yeast runs counter to previous studies in Escherichia coli and for the first time links thioredoxin with the redox state of glutathione in vivo.

A redox-dependent function of thioredoxin is necessary to sustain a rapid rate of DNA synthesis in yeast.

DNA replication is impaired in mutants of Saccharomyces cerevisiae which lack the two thioredoxin genes TRX1 and TRX2. Trx1p supports a normal rate of DNA replication only if the active site contains the redox active cysteines. Two mutant forms of Trx1p, one containing a Cys30Ser mutation and a second containing the Cys30Ser mutation in combination with a Cys33Ser mutation, were unable to sustain normal rates of DNA synthesis. The thioredoxin active-site mutants completed a round of replication in 66 min as opposed to 18 min observed for an isogenic wild type culture. Western blot analysis, using antibody generated against purified 6 x His-tagged Trx1p, showed that both mutant forms of Trx1p were present at the same levels as the wild-type protein. Thus the inability of the mutant proteins to promote DNA synthesis is not caused by degradation or poor expression, but rather by the loss of their reductive capacity. The results show that an optimal rate of DNA synthesis requires a redox function of thioredoxin. Since the measured levels of deoxyribonucleotides are normal in the thioredoxin mutants, thioredoxin either participates with ribonucleotide reductase in channeling a small subset of deoxyribonucleotides to sites of replication, or thioredoxin reduces and thereby activates an unidentified component of the replication machinery.

Deoxyribonucleotides are maintained at normal levels in a yeast thioredoxin mutant defective in DNA synthesis.

Deletion of both thioredoxin genes TRX1 and TRX2 of Saccharomyces cerevisiae reduces the rate of DNA replication. This observation, originally determined by flow cytometry, was confirmed by radiochemical labeling of synchronized cultures. Since thioredoxin is a hydrogen donor to ribonucleotide reductase, a priori the inhibition of DNA synthesis was predicted to be caused by a reduction in the deoxyribonucleotide pools. However, the levels of TTP, dCTP, dATP, and dGTP were either unchanged or slightly greater in the thioredoxin mutant (3.2, 0.91, 1.4, and 1.21 pmol/10(6) cells, respectively) versus the wild-type culture (2.5, 0.91, 1.0, and 0.68 pmol/10(6) cells, respectively). An impact on ribonucleotide reduction was seen by an increased accumulation of RNR1 and RNR2 transcripts in the thioredoxin mutant (4.3- and 6.8-fold, respectively). Increased RNR expression did not reflect a general response of the DNA replication machinery. POL1 (DNA polymerase I) and CDC8 (thymidylate kinase) transcription were unaltered, while histone H2B transcripts actually decreased by half. Two alternative models incorporating these results are discussed. One suggests that thioredoxin reduces a multiprotein complex channeling nucleotides to the replication apparatus. The second proposes that thioredoxin regulates the tempo of DNA replication directly by activating a component of the replication machinery.

The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae.

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.

A dosage-dependent suppressor of a temperature-sensitive calmodulin mutant encodes a protein related to the fork head family of DNA-binding proteins.

The cmd1-1 mutation of calmodulin causes temperature-sensitive growth in Saccharomyces cerevisiae. We have isolated a dosage-dependent suppressor of cmd1-1, designated HCM1. Twentyfold overexpression of HCM1 permits strains carrying cmd1-1 to grow at temperatures up to and including 34 degrees C but does not suppress the lethality of either cmd1-1 at higher temperatures or the deletion of CMD1. Thus, overexpression of HCM1 does not bypass the requirement for calmodulin but enhances the ability of the mutant calmodulin to function. HCM1 is not essential for growth, but deletion of HCM1 exacerbates the phenotype of a strain carrying cmd1-1. HCM1 is located on chromosome III, which was recently sequenced. Our results correct errors in the published DNA sequence. The putative polypeptide encoded by HCM1 is 564 amino acids long and has a predicted molecular weight of 63,622. Antisera prepared against Hcm1p detect a protein that is overproduced in yeast strains overexpressing HCM1 and has an apparent molecular mass of 65 kDa. Eighty-six amino acid residues in the N terminus of Hcm1p show 50% identity with a DNA-binding region of the fork head family of DNA-binding proteins. When fused to the DNA-binding domain of Gal4p, residues 139 to 511 of Hcm1p can act as a strong activator of transcription. However, overexpression of HCM1 does not affect the expression of calmodulin. Furthermore, Hcm1p does not bind to calmodulin in a gel overlay assay. Thus, overexpression of HCM1 enhances calmodulin function by an apparently indirect mechanism.

Thioredoxin genes in Saccharomyces cerevisiae: map positions of TRX1 and TRX2.

The two genes encoding thioredoxins in Saccharomyces cerevisiae, TRX1 and TRX2, map to chromosome XII and VII, respectively. From the DNA sequence of the intragenic region TRX1 is 500 bp downstream of PDC1. Tetrad analysis places TRX2 1.1 cM from ADE3, while a physical map of this region positions TRX2 4.5 kb downstream of ADE3. The mapping of TRX1 adjacent to PDC1 clarifies previous results (Muller, E. G. D. J. Biol. Chem. 266, 9194-9202, 1991) that suggested a third thioredoxin gene.

Thioredoxin deficiency in yeast prolongs S phase and shortens the G1 interval of the cell cycle.

Two thioredoxin genes from the yeast Saccharomyces cerevisiae were cloned using synthetic oligonucleotide probes. The DNA sequences of the two genes were found to be 74% identical. The two genes, designated TRX1 and TRX2, were mutagenized in vitro and used to construct a set of thioredoxin deletion mutants. The loss of either thioredoxin gene alone has no effect on cell growth or morphology. However, the simultaneous deletion of both thioredoxin genes profoundly affects the cell cycle. S phase is 3-fold longer, and G1 is virtually absent. In addition, the thioredoxin double mutant shows a 33% increase in generation time, a significant increase in cell size, and a greater proportion of large budded cells. The results suggest that in the absence of TRX1 and TRX2, a slow rate of DNA replication inhibits the normal progress of cellular reproduction. Surprisingly, the loss of both thioredoxins also leads to methionine auxotrophy. Thus yeast glutaredoxin is unable to substitute for thioredoxin in sulfate assimilation. As a first step in studying the cell cycle control mechanisms that respond to the thioredoxin deficiency, it was shown that cell viability does not require the function of RAD9, a known cell cycle checkpoint.

Thioredoxin is essential for photosynthetic growth. The thioredoxin m gene of Anacystis nidulans.

We have taken advantage of the transformation properties of the cyanobacterium Anacystis nidulans R2 to investigate the importance of thioredoxin for photosynthetic growth. The gene encoding thioredoxin m, designated trxM, was cloned from A. nidulans using a synthetic oligonucleotide probe. Based on the nucleotide sequence, thioredoxin m of A. nidulans is composed of 107 amino acids and shares 84, 48, and 48% sequence identity with thioredoxins from Anabaena, spinach, and Escherichia coli, respectively. The trxM gene is single copy and is transcribed on a 510-nucleotide mRNA. We demonstrate that disruption of the trxM gene with a kanamycin resistance gene cartridge is a lethal mutation. Although dispensable in E. coli, thioredoxin is essential for the photosynthetic growth of A. nidulans.

Sponge cell aggregation.

Dissociated sponge cell system has proved to be a useful model to study the process of cell aggregation both on cellular and subcellular level. The purpose of this review is to discuss recent results obtained from experiments with the marine sponge Geodia cydonium. Dissociated cells form functional aggregates during a process which can be sub-divided into three phases: first, formation of small primary aggregates in the presence of Ca2+; second, formation of secondary aggregates in the presence of an aggregation factor and third, reconstitution of a functional system of water-containing channels by rearrangement in the secondary aggregates. On subcellular level a series of macromolecules are known which are involved in the control of aggregation and separation of sponge cells: Aggregation factor, aggregation receptor, anti-aggregation receptor, beta-glucuronidase, beta-glucuronosyltransferase, beta-galactosyltransferase, beta-galactosidase and a lectin. These components might be linked in the following sequence: (a) Activation of the aggregation receptor by its enzymic glucuronylation; (b) Adhesive recognition of the cells, mediated by the aggregation factor and the glucuronylated aggregation receptor; (c) Inactivation of the aggregation receptor by its deglucuronylation with the membrane-associated beta-glucuronidase; (d) Cell separation due to either the loss of the recognition site (glucuronic acid) of the aggregation receptor for the aggregation factor or to an inactivation of the aggregation factor by the anti-aggregation receptor. The activity of the anti-aggregation receptor is most likely controlled by the Geodia lectin. The events leading to cell-cell recognition cause a change in the following metabolic events: Increase of oxygen uptake, decrease of cyclic AMP level, increase of cyclic GMP level and stimulation of programmed syntheses.