Conditions Of Repression Research Articles

English

  Cellulose degrading organisms have been used for the conversion of cellulolytic materials into soluble sugars or solvents in several biotechnological and industrial applications. In this report, a mutant of Bacillus pumilus was obtained after chemical mutagenesis and screened for cellulase production. This mutant named BpCRI 6 was selected for its ability to produce cellulase under catabolite repression. Cellulase yield by BpCRI 6 was four times higher than that of the wild type under optimum growth conditions (pH 6.5, 25°C and Ca2+ 1mM). In shaking flask cultures, production of cellulase  by the wild type was completely repressed in the presence of 25 mM glucose, while BpCRI 6 strain still exhibited a residual cellulase production of 80 and 40% at 25 mM and 40 mM of glucose concentrations respectively. The mutant strain is stable and grows rapidly in liquid and solid media. Under conditions of catabolite repression (40 mM of glucose), the production of cellulase by this mutant is particularly significant when compared to Trichoderma reesei a well-known cellulase producer, which is under control of end-product inhibition. This is the first report of a successful catabolite repression insensitivity of cellulase production by a mutant of B. pumilus.   Key words: Cellulase, Bacillus pumilus, BpCRI 6, Catabolite repression.

Targeted cytosine methylation for in vivo detection of protein-DNA interactions.

We report a technique, named targeted gene methylation (TAGM), for identifying in vivo protein-binding sites in chromatin. M.CviPI, a cytosine-5 DNA methyltransferase recognizing GC sites, is fused to a DNA-binding factor enabling simultaneous detection of targeted methylation, factor footprints, and chromatin structural changes by bisulfite genomic sequencing. Using TAGM with the yeast transactivator Pho4, methylation enrichments of up to 34- fold occur proximal to native Pho4-binding sites. Additionally, significant selective targeting of methylation is observed several hundred nucleotides away, suggesting the detection of long-range interactions due to higher-order chromatin structure. In contrast, at an extragenic locus lacking Pho4-binding sites, methylation levels are at the detection limit at early times after Pho4 transactivation. Notably, substantial amounts of methylation are targeted by Pho4-M.CviPI under repressive conditions when most of the transactivator is excluded from the nucleus. Thus, TAGM enables rapid detection of DNA-protein interactions even at low occupancies and has potential for identifying factor targets at the genome-wide level. Extension of TAGM from yeast to vertebrates, which use methylation to initiate and propagate repressed chromatin, could also provide a valuable strategy for heritable inactivation of gene expression.

Sigma 54 levels and physiological control of the Pseudomonas putida Pu promoter.

The cellular levels of the alternative sigma factor sigma(54) of Pseudomonas putida have been examined in a variety of growth stages and culture conditions with a single-chain Fv antibody tailored for detection of scarce proteins. The levels of sigma(54) were also monitored in P. putida strains with knockout mutations in ptsO or ptsN, known to be required for the C-source control of the sigma(54)-dependent Pu promoter of the TOL plasmid. Our results show that approximately 80 +/- 26 molecules of sigma(54) exist per cell. Unlike that in relatives of Pseudomonas (e.g., Caulobacter), where fluctuations of sigma(54) determine adaptation and differentiation when cells face starvation, sigma(54) in P. putida remains unexpectedly constant at different growth stages, in nitrogen starvation and C-source repression conditions, and in the ptsO and ptsN mutant strains analyzed. The number of sigma(54) molecules per cell in P. putida is barely above the predicted number of sigma(54)-dependent promoters. These figures impose a framework on the mechanism by which Pu (and other sigma(54)-dependent systems) may become amenable to physiological control.

Development of a reporter system for the yeast Schwanniomyces occidentalis: influence of DNA composition and codon usage.

In this paper we report on searching for suitable reporters to monitor gene expression and protein secretion in the amylolytic yeast Schwanniomyces occidentalis. Several potential reporter and marker genes, formerly shown to be functional in other yeasts, were cloned downstream from the homologous invertase gene (INV) promoter and their activity was followed in conditions of repression and derepression of the INV promoter. However, neither beta-glucuronidase nor beta-lactamase nor phleomycin resistance-conferring gene, all originating from E. coli, were expressed in S. occidentalis cells to such a level to allow for monitoring of their activity. All the reporter genes tested have a higher percentage of GC (47-62%) in their DNA compared to the DNA composition of S. occidentalis genes that are more AT-rich (36% GC). The codon usage of all the reporter genes also varies from that of 16 so far sequenced S. occidentalis genes. This suggests that an appropriate composition of DNA and a codon usage similar to S. occidentalis genes might be very important parameters for an efficient expression of a heterologous gene in Schwanniomyces occidentalis. Indeed, two genes originating from Staphylococcus aureus, with an AT-content in their DNA similar to that of S. occidentalis, were functionally expressed in S. occidentalis cells. Both a phleomycin resistance-conferring gene and a chloramphenicol acetyltransferase-encoding gene thus represent suitable reporters of gene expression and protein secretion in S. occidentalis. Additionally, we show in this work that the transcription-regulating region and the signal peptide sequence of the S. occidentalis invertase gene were efficient to direct gene expression and subsequent protein secretion in Saccharomyces cerevisiae.

Stress-induced gene expression requires programmed recovery from translational repression.

Active repression of protein synthesis protects cells against protein malfolding during endoplasmic reticulum stress, nutrient deprivation and oxidative stress. However, long-term adaptation to these conditions requires synthesis of new stress-induced proteins. Phosphorylation of the alpha-subunit of translation initiation factor 2 (eIF2alpha) represses translation in diverse stressful conditions. GADD34 is a stress-inducible regulatory subunit of a holophosphatase complex that dephosphorylates eIF2alpha, and has been hypothesized to play a role in translational recovery. Here, we report that GADD34 expression correlated temporally with eIF2alpha dephosphorylation late in the stress response. Inactivation of both alleles of GADD34 prevented eIF2alpha dephosphorylation and blocked the recovery of protein synthesis, normally observed late in the stress response. Furthermore, defective recovery of protein synthesis markedly impaired translation of stress-induced proteins and interfered with programmed activation of stress-induced genes in the GADD34 mutant cells. These observations indicate that GADD34 controls a programmed shift from translational repression to stress-induced gene expression, and reconciles the apparent contradiction between the translational and transcriptional arms of cellular stress responses.

Interaction of NF-E2 in the Human β-Globin Locus Control Region before Chromatin Remodeling

When transcription is initiated under repressive conditions, such as when chromatin are packed together, binding followed by the functioning of key components in the transcriptional apparatus should be appropriately facilitated in the chromatin architecture. We provide evidence that the erythroid-specific enhancer- binding protein NF-E2 interacts with the cognate motif at DNase I-hypersensitive site 2 of the human beta-globin locus control region in a repressive state. The nucleosome containing the NF-E2-binding site showed characteristic rotational and translational phases in vitro. The binding site had less affinity to the histone octamers than nearby regions while showing greater accessibility to DNase I and micrococcal nuclease. Furthermore, the motif was recognized by the exogenous NF-E2 protein expressed in HeLa cells, which have a repressive state of chromatin at the beta-globin locus, as shown by ligation-mediated PCR and chromatin immunoprecipitation assay. These lines of evidence indicate that NF-E2 interacts with the cognate motif on the nucleosome before chromatin is remodeled.

The regulatory cascade that activates the Hrp regulon in Erwinia herbicola pv. gypsophilae.

The pathogenicity of Erwinia herbicola pv. gypsophilae (Ehg) is dependent on a plasmid (pPATH(Ehg)) that harbors the hrp gene cluster and additional virulence genes. The hrp regulatory cascade of Ehg comprises an hrpXY operon encoding a two-component system; hrpS encoding a transcriptional factor of the NtrC family and hrpL encoding an alternative sigma factor. Results obtained suggest the following signal transduction model for activating the Hrp regulon: phosphorylated HrpY activates hrpS, HrpS activates hrpL, and HrpL activates genes containing "hrp box" promoter. This model was supported by studies on the effects of mutations in the regulatory genes on pathogenicity and complementation analysis. Nonpolar mutations in hrpX did not affect virulence or transcription of downstream genes. Site-directed mutagenesis of the conserved aspartate 57 in HrpY suggested that its phosphorylation is crucial for activating the hrp regulatory cascade. Studies on the effects of mutations in the hrp regulatory genes on transcriptional activity of downstream genes or of their isolated promoters in planta showed dependency of hrpS expression on active HrpY, of hrpL expression on active HrpS, and of hrpN or hrpJ expression on active HrpL. These results were also partially supported by overexpression of regulatory genes under in vitro conditions. The hrpXY is constitutively expressed with high basal levels under repressive conditions, in contrast to hrpS and hrpL, which exhibit low basal expression levels and are environmentally regulated.

Cultural Production in Context

The Art of Transition, as its title indicates, offers an analysis of a broad scope of cultural production as it moves from conditions of dictatorship to democracy in the context of neoliberalism and globalization. It focuses primarily on the cultural production of Argentina and Chile, mainly in the fields of fictional narrative and poetry, though some references to the visual arts are included. Francine Masiello's book, without any doubt, is a complex, inspiring, and a very timely piece of work. In fact, it is undeniable that artand the process of creation and creativity it involves constitutes a key cultural artifact and form of cultural expression of any society. Also undeniable is the strong relationship between Latin American literary discourses and the sociohistorical context in which they are produced. This is clear, for instance, in the literature produced under repressive conditions in South America, which could be considered as a cultural response to dictatorship and authoritarianism. Likewise, given the dramatic economic and cultural changes we are experiencing under the aegis of neoliberalism and globalization, it seems imperative, now more than ever, to consider the relationship between cultural production and the socio-historical context, as well as that between the dominant mode of production and the mode of production of a literary text or a piece of art. For instance, one can consider some of the more recent literary production which, in some cases, seems to be strongly conditioned by the market and cultural industry policies, becoming little more than rhetoric for mass consumption. However, along with this kind of cultural production, there is another kind of literary expressions that doesn't forget that literature is a form of art and aesthetic experience, as much as it is a cultural expression of resistance to the market as the prevailing orientation.

Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates

BackgroundA rapid microtiter plate based sandwich hybridization assay was developed for detection and quantification of single RNA species using magnetic beads. Following solution hybridization target RNA molecules were collected by biotin-streptavidin affinity binding and detected by fluorescence signal generated by alkaline phosphatase. The 18S rRNA and SUC2 mRNA of Saccharomyces cerevisiae were used as model RNA target molecules.ResultsThe sensitivity of the assay was approximately 1.2 × 109 (2 fmol) molecules of target RNA. The developed method was feasible with crude cell lysates of S. cerevisiae carlsbergensis and was evaluated by measuring the levels of 18S rRNA during cell growth and SUC2 mRNA under repressive and inductive conditions. The 18S rRNA expression level followed the changes in the specific growth rate. SUC2 mRNA levels were in good correlation with the measured invertase enzyme activities.ConclusionsThe here presented sandwich hybridisation method was succefully applied for monitoring the amounts of ribosomal RNA and mRNA with high expression level in shake flask cultivation conditions. Sandwich hybridisation method offers a fast and convenient tool for following single key RNA species of interest in the production conditions.

Footsteps of Red Ink: Body and Landscape in "Lolly Willowes"

had spent the afternoon field, of unusual form, for it was triangular (Warner, Willowes 141). The date is August 1922, the place is near the fictional village of Great Mop Buckinghamshire, and Laura is Laura Erminia Willowes (called by relatives), protagonist of Sylvia Townsend Warner's 1926 novel Willowes. Laura tramps around the field, turning savagely when she came to the edge, because escape from imperialist patriarchy, brother's family, has been foiled. Into Great Mop, an idyllic country village which Laura sought solitary independence, has come assuming nephew Titus. Dogging steps (Where are you off to, Aunt Lolly? Wait minute, and I'll come too [142]), Titus threatens to return Laura to her old employment of being Aunt Lolly (149)--that is, an aging spinster aunt residing London with patronizing brother and his family. With Titus's arrival, the green and pleasant land around Great Mop has become distinctly unpleasant, just as organized by an English social and sexual order as are the more obviously man-made environs of London that Laura sought to escape. Laura paces the boundaries of the in despair and rebellion (142), unable to envision solution that will free from the airless domestic spaces offered by the interlocking claims of class, gender, and presumptive heterosexuality. She finally calls out, No! You shan't get me. I won't go back. I won't.... Oh! Is there no help? (151). And into the novel sails Satan, figured here as a kind of black knight, wandering about and succoring decayed gentlewomen (211), to offer Laura the freedom of the manor exchange for commitment to witchcraft. Willowes is increasingly studied as Warner's academic profile rises, (1) but the events following the carefully showcased speech act above still puzzle critics and readers. Why should Satan respond to this wishful call, offering to dispatch Titus from Great Mop and guarantee Laura's safety? Why should she make pact with this masculine Master (210), when so much of the novel is about finding space where female sexuality is free from male control? (2) And why should this novel take such seemingly whimsical turn to the fantastic? A partial answer may be found that of unusual form Laura paces, for Warner's novel is commentary on the semiotics and politics of landscape as structuring agent subjectivity. The sentence begins as pure representation: country walks fields, however unsatisfying, do not arouse hermeneutic suspicion. Repeating the word the second clause, however, Warner balances it with form, alliteratively cinching together the natural field with its scientific meanings. In the final clause, another alliterated f for encourages the correlation between the natural and the abstract by giving the geometrical description, triangular. The language Warner uses enforces the connection between the material and the linguistic, engaging the purportedly material field semiotic system endowed with substantial organizational power. The landscape, like language, calculably structures reality instead of merely being. Thus Laura, tired, stumbl[ing], walk[ing] slowly, and bitter (141-43), is involved more than an agon with Titus; she struggles against of forms designed to subject bodily to their purposes. Reflecting character's inner conflict the setting is common literary practice, but Warner is not simply reflecting: she makes the landscape material presence, an agent Laura's struggle for place. (3) This moment, showcasing Laura's anxious, uncomfortable body, begins transformation from an individual struggling with particular set of familial conditions to an embodied subject (Grosz 22; emphasis omitted) whose agency is produced by relationship with spaces replete with ideological significance. Willowes is not only about disembodied ego escaping from sexually repressive conditions to reach an abstract freedom--as Laura had thought--but about whole classes of subjected bodies corralled by an ideology of place naturalized through historical and literary precedent. …

Footsteps of Red Ink: Body and Landscape in Lolly Willowes

"Laura had spent the afternoon in a field, a field of unusual form, for it was triangular" (Warner, Lolly Willowes 141). The date is August 1922, the place is near the fictional village of Great Mop in Buckinghamshire, and Laura is Laura Erminia Willowes (called Lolly by her relatives), protagonist of Sylvia Townsend Warner's 1926 novel Lolly Willowes. Laura tramps around the field, "turning savagely when she came to the edge," because her escape from imperialist patriarchy, embodied in her brother's family, has been foiled. Into Great Mop, an idyllic country village in which Laura sought solitary independence, has come her assuming nephew Titus. Dogging her steps ("Where are you off to, Aunt Lolly? Wait a minute, and I'll come too" [142]), Titus threatens to return Laura to "her old employment of being Aunt Lolly" (149)--that is, an aging spinster aunt residing in London with her patronizing brother and his family. With Titus's arrival, the green and pleasant land around Great Mop has become distinctly unpleasant, just as organized by an English social and sexual order as are the more obviously man-made environs of London that Laura sought to escape. Laura paces the boundaries of the field "in despair and rebellion" (142), unable to envision a solution that will free her from the airless domestic spaces offered her by the interlocking claims of class, gender, and presumptive heterosexuality. She finally calls out, "No! You shan't get me. I won't go back. I won't.... Oh! Is there no help?" (151). And into the novel sails Satan, figured here as "a kind of black knight, wandering about and succoring decayed gentlewomen" (211), to offer Laura the freedom of the manor in exchange for her commitment to witchcraft. Lolly Willowes is increasingly studied as Warner's academic profile rises, (1) but the events following the carefully showcased speech act above still puzzle critics and readers. Why should Satan respond to this wishful call, offering to dispatch Titus from Great Mop and guarantee Laura's safety? Why should she make a pact with this masculine "Master" (210), when so much of the novel is about finding a space where female sexuality is free from male control? (2) And why should this novel take such a seemingly whimsical turn to the fantastic? A partial answer may be found in that "field of unusual form" Laura paces, for Warner's novel is a commentary on the semiotics and politics of landscape as a structuring agent in subjectivity. The sentence begins as pure representation: country walks in fields, however unsatisfying, do not arouse hermeneutic suspicion. Repeating the word field in the second clause, however, Warner balances it with form, alliteratively cinching together the natural "field" with its scientific meanings. In the final clause, another alliterated f in for encourages the correlation between the natural and the abstract by giving the field a geometrical description, triangular. The language Warner uses enforces the connection between the material and the linguistic, engaging the purportedly material "field" in a semiotic system endowed with substantial organizational power. The landscape, like language, calculably structures reality instead of merely "being." Thus Laura, "tired," "stumbl[ing]," "walk[ing] slowly," and bitter (141-43), is involved in more than an agon with Titus; she struggles against a field of forms designed to subject her bodily to their purposes. Reflecting a character's inner conflict in the setting is a common literary practice, but Warner is not simply reflecting: she makes the landscape a material presence, an agent in Laura's struggle for a place. (3) This moment, showcasing Laura's anxious, uncomfortable body, begins her transformation from an individual struggling with a particular set of familial conditions to an "embodied" subject (Grosz 22; emphasis omitted) whose agency is produced by her relationship with spaces replete with ideological significance. Lolly Willowes is not only about a disembodied ego escaping from sexually repressive conditions to reach an abstract freedom--as Laura had thought--but about whole classes of subjected bodies corralled by an ideology of place naturalized through historical and literary precedent. …

Contemporary European migrations, civic stratification and citizenship

Since the late 1980s there has been a diversification of European migratory flows. States, which remain the key actors in migration policies despite growing European harmonisation, have responded to these complex patterns and contradictory pressures by diversifying migrant categories and statuses. The heterogeneity of migrant statuses, especially the growth in the number of those on temporary statuses and the undocumented, challenges the thesis of a post national citizenship as having been achieved in relation to non European Union or third country migrants in the European Union. The recent proposals to extend mobility and socio-economic rights to third country nationals in the EU will only apply to long-term residents with evidence of adequate resources. The inability to confront the contradictions and tensions between economic needs and political closure will lead to more repressive conditions for undocumented migrants and reinforced border controls, especially in the light of the electoral success of Far Right parties and widespread anti-immigrant attitudes in a number of European Union states.

Reconstitution of Nucleosome Positioning, Remodeling, Histone Acetylation, and Transcriptional Activation on the PHO5Promoter

The PHO5 gene promoter is an important model for the study of gene regulation in the context of chromatin. Upon PHO5 activation the chromatin structure is reconfigured, but the mechanism of this transition remains unclear. Using templates reconstituted into chromatin with purified recombinant yeast core histones, we have investigated the mechanism of chromatin structure reconfiguration on the PHO5 promoter, a prerequisite for transcriptional activation. Footprinting analyses show that intrinsic properties of the promoter DNA are sufficient for translational nucleosome positioning, which approximates that seen in vivo. We have found that both Pho4p and Pho2p can bind their cognate sites on chromatin-assembled templates without the aid of histone-modifying or nucleosome-remodeling factors. However, nucleosome remodeling by these transcriptional activators requires an ATP-dependent activity in a yeast nuclear extract fraction. Finally, transcriptional activation on chromatin templates requires acetyl-CoA in addition to these other activities and cofactors. The addition of acetyl-CoA results in significant core histone acetylation. These findings indicate that transcriptional activation requires Pho4p, Pho2p, nucleosome remodeling, and nucleosome acetylation. Furthermore, we find that DNA binding, nucleosome remodeling, and transcriptional activation are separable steps, facilitating biochemical analysis of the PHO5 regulatory mechanism.

CAMP-induced modulation of the growth yield of Saccharomyces cerevisiae during respiratory and respiro-fermentative metabolism

The aim of this study was to investigate the effects of an overactivation of the cAMP/protein kinase A signaling pathway on the energetic metabolism of growing yeast. By using a cAMP-permeant mutant strain, we show that the rise in intracellular cAMP activates both anabolic and catabolic pathways. Indeed, different physiological patterns were observed with respect to the growth condition: (i) When cells were grown with a limiting amount of lactate, cAMP addition markedly increased the growth rate, whereas it only slightly increased the mitochondrial and cellular protein content. In parallel, the respiratory rate increased and the growth yield, as assessed by direct microcalorimetry, was not significantly modified by cAMP. (ii) Under conditions where the growth rate was already optimal (high lactate concentration), exogenous cAMP led to a proliferation of well-coupled mitochondria within cells and to an accumulation of cellular and mitochondrial proteins. This phenomenon was associated with a rise in the respiratory activity, thus leading to a drop in the growth yield. (iii) Under conditions of catabolic repression (high glucose concentration), cAMP addition markedly increased the fermentation rate and decreased the growth yield. It is concluded that overactivation of the cAMP/PKA pathway leads to uncoupling between biomass synthesis and catabolism, under conditions where an optimal growth rate is sustained by either a fermentative or a respiratory metabolism.

Political Repression and Public Perceptions of Human Rights

Based on data from 18 Central and East European countries collected between 1991 and 1996, this article tests informational assumptions underlying strategic interaction and collective action models of goverment repression and dissent. Specifically, we investigate whether citizens' perceptions of human rights conditions in a country are systematically related to that country's actual conditions of government repression. The analysis suggests that there is a significant relationship between evalutions of human rights conditions and levels of government repression. Moreover, it shows that other political and economic conditions affect human rights evaluations, but that these relations do not lead to a weaening in the relationship between repressive conditions and public perceptions of human rights.

Process technological effects of deletion and amplification of hydrophobins I and II in transformants of Trichoderma reesei.

Transformants of the Trichoderma reeseistrains QM9414 and Rut-C30 were constructed in which the genes for the two major hydrophobin proteins, hydrophobins I (HFBI) and II (HFBII), were deleted or amplified by molecular biological techniques. Growth parameters and foam production of the transformant strains were compared with the corresponding properties of the parent strains by cultivation in laboratory bioreactors under conditions of catabolite repression (glucose medium) or induction of cellulolytic enzymes and other secondary metabolites (cellulose and lactose media). All the transformed strains exhibited vegetative growth properties similar to those of their parent. The Delta hfb2 (but not the Delta hfb1) transformant showed reduced tendency to foam, whereas both strains overproducing hydrophobins foamed extensively, particularly in the case of HFBII. Enzyme production on cellulose medium was unaltered in the Delta hfb2 transformant VTT D-99676, but both the Delta hfb2 and HFBII-overproducing transformants exhibited somewhat decreased enzyme production properties on lactose medium. Production of HFBI by the multi-copy transformant VTT D-98692 was almost 3-fold that of the parent strain QM9414. Overproduction of HFBII by the transformant VTT D-99745, obtained by transformation with three additional copies of the hfb2 gene under the cbh1 promoter, was over 5-fold compared to production by the parent strain Rut-C30. The Delta hfb2transformant VTT D-99676 produced a greatly increased number of spores on lactose medium compared with the parent strain, whereas the HFBII-overproducing transformant VTT D-99745 produced fewer spores.

Coregulation of Alternative Transcripts of the STA2 Gene in the Yeast Saccharomyces cerevisiae

It is known that upon STA2 gene expression in Saccharomyces cerevisiae cells, two transcripts of different lengths are formed. These fragments contain different start codons of translation (AUG1 and AUG2) located in the same reading frame. The ratio between expression levels of proteins translated from AUG1 and AUG2 was invariably about 2 : 7 and did not depend on the choice of the reporter product (secreted glucoamylase (GA) or β-galactosidase accumulated in cells). Neither this ratio depended on glucose repression/derepression. Based on the assumed proportional relationship between levels of transcription and translation of the STA2 gene in yeast cells, all these results cumulatively indicate that the two STA2 transcripts are coregulated. The production of secreted GA was also shown to be markedly stimulated at the posttranscriptional level under conditions of glucose repression.

Regulation of carbon utilization by sulfur availability in Escherichia coli and Salmonella typhimurium.

Different pleiotropic transcriptional regulators are known to function in the coordination of regulons concerned with carbon, nitrogen, sulfur, phosphorus and iron metabolism, but how expression profiles of these different regulons are coordinated with each other is not known. The basis for the effects of cysB mutations on carbon utilization in Escherichia coli and Salmonella typhimurium was examined. cysB mutations affected the utilization of some carbon sources more than others and these effects could be partially, but not completely, reversed by the inclusion of cysteine or djenkolate in the growth medium. Assays of transport systems and enzymes concerned with glucitol and alanine utilization showed that these activities were depressed in cysB mutants relative to isogenic wild-type strains, and cysteine or djenkolate present in the growth media partially restored these activities. Using transcriptional fusions to the fdo (formate dehydrogenase) and gut (glucitol) operons, it was shown that decreased expression resulted from defects at the transcriptional level. Furthermore, the effects of loss of CysB were much less pronounced under conditions of catabolite repression than in the absence of a catabolite-repressing carbon source, and cAMP largely reversed the effect of the loss of CysB. Comparable effects were seen for E. coli lacZ gene expression under the control of its own native promoter, and sulfur limitation in a cysB mutant depressed net cAMP production in a cAMP phosphodiesterase mutant. Adenylate cyclase thus appears to be responsive to sulfur deprivation. These observations may have physiological significance allowing carbon and sulfur regulon coordination during the growth of enteric bacteria in response to nutrient availability.

Environmental Regulation of Lateral Root Initiation in Arabidopsis

Plant morphology is dramatically influenced by environmental signals. The growth and development of the root system is an excellent example of this developmental plasticity. Both the number and placement of lateral roots are highly responsive to nutritional cues. This indicates that there must be a signal transduction pathway that interprets complex environmental conditions and makes the "decision" to form a lateral root at a particular time and place. Lateral roots originate from differentiated cells in adult tissues. These cells must reenter the cell cycle, proliferate, and redifferentiate to produce all of the cell types that make up a new organ. Almost nothing is known about how lateral root initiation is regulated or coordinated with growth conditions. Here, we report a novel growth assay that allows this regulatory mechanism to be dissected in Arabidopsis. When Arabidopsis seedlings are grown on nutrient media with a high sucrose to nitrogen ratio, lateral root initiation is dramatically repressed. Auxin localization appears to be a key factor in this nutrient-mediated repression of lateral root initiation. We have isolated a mutant, lateral root initiation 1 (lin1), that overcomes the repressive conditions. This mutant produces a highly branched root system on media with high sucrose to nitrogen ratios. The lin1 phenotype is specific to these growth conditions, suggesting that the lin1 gene is involved in coordinating lateral root initiation with nutritional cues. Therefore, these studies provide novel insights into the mechanisms that regulate the earliest steps in lateral root initiation and that coordinate plant development with the environment.

Gamma-Glutamyl transpeptidase in the yeast Saccharomyces cerevisiae and its role in the vacuolar transport and metabolism of glutathione.

In the yeast Saccharomyces cerevisiae, the enzyme gamma-glutamyl transpeptidase (gamma-GT; EC 2.3.2.2) is a glycoprotein that is bound to the vacuolar membrane. The kinetic parameters of GSH transport into isolated vacuoles were measured using intact vacuoles isolated from the wild-type yeast strain Sigma 1278b, under conditions of gamma-GT synthesis (nitrogen starvation) and repression (growth in the presence of ammonium ions). Vacuoles devoid of gamma-GT displayed a K(m) (app) of 18+/-2 mM and a V(max) (app) of 48.5+/-5 nmol of GSH/min per mg of protein. Vacuoles containing gamma-GT displayed practically the same K(m), but a higher V(max) (app) (150+/-12 nmol of GSH/min per mg of protein). Vacuoles prepared from a disruptant lacking gamma-GT showed no increase in V(max) (app) with nitrogen starvation. From a comparison of the transport data obtained for vacuoles isolated from various reference and mutant strains, it appears that the yeast cadmium factor 1 (YCF1) transport system accounts for approx. 70% of the GSH transport capacity of the vacuoles, the remaining 30% being due to a vacuolar (H(+)) ATPase-coupled system. The V(max) (app)-increasing effect of gamma-GT concerns only the YCF1 system. gamma-GT in the vacuolar membrane activates the Ycf1p transporter, either directly or indirectly. Moreover, GSH accumulating in the vacuolar space may exert a feedback effect on its own entry. Excretion of glutamate from radiolabelled GSH in isolated vacuoles containing gamma-GT was also measured. It is proposed that gamma-GT and a L-Cys-Gly dipeptidase catalyse the complete hydrolysis of GSH stored in the central vacuole of the yeast cell, prior to release of its constitutive amino acids L-glutamate, L-cysteine and glycine into the cytoplasm. Yeast appears to be a useful model for studying gamma-GT physiology and GSH metabolism.