Effect Of Derepression Research Articles

T7-lac promoter vectors spontaneous derepression caused by plant-derived growth media may lead to serious expression problems: a systematic evaluation

BackgroundThe widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. Various factors can strongly affect the successful construction and stable maintenance of clones and the resulting biosynthesis levels. These include an appropriate selection of recombinant hosts, expression systems, regulation of promoters, the repression level at an uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics, the presence/absence of chaperons and others. However, optimization of the growth medium’s composition is often overlooked. We systematically evaluate this factor, which can have a dramatic effect on the expression of recombinant proteins, especially those which are toxic to a recombinant host.ResultsCommonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed Open Reading Frames (ORFs) with a wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28—which codes for thermophilic endolysin (moderately toxic); and (iii) tthHB27IRM—which codes for thermophilic restriction endonuclease-methyltransferase (REase-MTase)—RM.TthHB27I (very toxic). The use of plant-derived peptones (soy peptone and malt extract) in a culture medium causes the T7-lac expression system to leak. We show that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of the culture, the stability of clones and biosynthesis levels.ConclusionsThe use of plant-derived peptones in a culture medium when using T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins which are toxic to a recombinant host, this can result in mutations or deletions in the expression vector and/or cloned gene, the death of the host or highly decreased expression levels. This phenomenon is caused by the content of certain saccharides in plant peptones, some of which (galactosides) may act as T7-lac promoter inducer by interacting with a Lac repressor. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or to use them with caution and perform a pilot-scale evaluation of the derepression effect on a case-by-case basis.

Rrp6 Regulates Heterochromatic Gene Silencing via ncRNA RUF6 Decay in Malaria Parasites.

The heterochromatin environment plays a central role in silencing genes associated with the malaria parasite's development, survival in the host, and transmission to the mosquito vector. However, the underlying mechanism regulating the dynamic chromatin structure is not understood yet. Here, we have uncovered that Plasmodium falciparum Rrp6, an orthologue of eukaryotic RNA exosome-associated RNase, controls the silencing of heterochromatic genes. PfRrp6 knockdown disrupted the singular expression of the GC-rich ncRNA RUF6 family, a known critical regulator of virulence gene expression, through the stabilization of the nascent transcripts. Mechanistic investigation showed that the accumulation of the multiple RUF6 ncRNAs triggered local chromatin remodeling in situ, which activated their adjacent var genes. Strikingly, chromatin isolation by RNA purification analysis (ChIRP-seq) revealed that a remarkable RUF6 ncRNA had interacted with distal heterochromatin regions directly and stimulated a global derepression effect on heterochromatic genes, including all variant gene families and the sexual commitment-associated regulator ap2-g gene. Collectively, Rrp6 appears to conduct the epigenetic surveillance of heterochromatic gene expression through controlling RUF6 levels, thereby securing antigenic variation and sexual commitment of malaria parasites during the infection of the host.IMPORTANCE Malaria remains a major public health and economic burden. The heterochromatin environment controls the silencing of genes associated with the fate of malaria parasites. Previous studies have demonstrated that a group of GC-rich ncRNAs (RUF6) is associated with the mutually exclusive expression of var genes, but the underlying mechanisms remain elusive. Here, through a series of genetic manipulation and genome-wide multiomics analysis, we have identified the plasmodial orthologue of RNA exosome-associated Rrp6 as an upstream regulator of RUF6 expression and revealed that the dysregulation of RUF6 upon Rrp6 knockdown triggered local chromatin alteration, thereby activating most heterochromatic genes via direct interaction of RUF6 and distal gene loci. This finding not only uncovered the in-depth mechanism of RUF6-mediated regulation of heterochromatic genes but also identified Rrp6 as a novel regulator of gene expression in human malaria parasites, which provides a new target for developing intervention strategies against malaria.

Genetic interactions and transcriptomics implicate fission yeast CTD prolyl isomerase Pin1 as an agent of RNA 3' processing and transcription termination that functions via its effects on CTD phosphatase Ssu72.

The phosphorylation pattern of Pol2 CTD Y1S2P3T4S5P6S7 repeats comprises an informational code coordinating transcription and RNA processing. cis–trans isomerization of CTD prolines expands the scope of the code in ways that are not well understood. Here we address this issue via analysis of fission yeast peptidyl-prolyl isomerase Pin1. A pin1Δ allele that does not affect growth per se is lethal in the absence of cleavage-polyadenylation factor (CPF) subunits Ppn1 and Swd22 and elicits growth defects absent CPF subunits Ctf1 and Dis2 and termination factor Rhn1. Whereas CTD S2A, T4A, and S7A mutants thrive in combination with pin1Δ, a Y1F mutant does not, nor do CTD mutants in which half the Pro3 or Pro6 residues are replaced by alanine. Phosphate-acquisition genes pho1, pho84 and tgp1 are repressed by upstream lncRNAs and are sensitive to changes in lncRNA 3′ processing/termination. pin1Δ hyper-represses PHO gene expression and erases the de-repressive effect of CTD-S7A. Transcriptional profiling delineated sets of 56 and 22 protein-coding genes that are down-regulated and up-regulated in pin1Δ cells, respectively, 77% and 100% of which are downregulated/upregulated when the cis-proline-dependent Ssu72 CTD phosphatase is inactivated. Our results implicate Pin1 as a positive effector of 3′ processing/termination that acts via Ssu72.

Effect of ampC derepression on cefepime MIC in Enterobacterales with chromosomally encoded inducible AmpC β-lactamase

ObjectivesWe aimed to evaluate the effect of ampC derepression on the cefepime MIC in different species of Enterobacterales with chromosomally encoded inducible AmpC β-lactamase. MethodsWe analysed a large number of wild-type/mutant pairs (n = 1030 in total). Cefepime MICs were determined by broth microdilution according to EUCAST recommendations. ResultsampC derepression led to increases in MIC by up to 10 dilutions, and significant increases by > 2 MIC dilutions were common across species (744/1030 mutants (72.2%) in total). Interestingly, the frequency of cefepime S→I/S→R transitions varied considerably between species: 66.3% in Enterobacter cloacae complex (167/252 mutants), 1.1% in Klebsiella aerogenes (2/180 mutants), 18.1% in Citrobacter freundii complex (50/277 mutants), 36.4% in Hafnia alvei (59/162 mutants), 19.0% in Providencia rettgeri (4/21 mutants), 22.9% in Providencia stuartii (11/48 mutants), 12.3% in Serratia marcescens (7/57 mutants), 20.0% in Serratia liquefaciens (6/30 mutants) and 0% in Morganella morganii (0/3 mutants). ConclusionsOur data show that the cefepime MIC is often increased by ampC derepression. However, the risk of S→I/S→R transition is dependent on the species.

H-NS Mutation-Mediated CRISPR-Cas Activation Inhibits Phage Release and Toxin Production of Escherichia coli Stx2 Phage Lysogen

Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. There is limited understanding of the effect that an Escherichia coli (E. coli) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immune system has on Stx phage lysogen. We investigated heat-stable nucleoid-structuring (H-NS) mutation-mediated CRISPR-Cas activation and its effect on E. coli Stx2 phage lysogen. The Δhns mutant (MG1655Δhns) of the E. coli K-12 strain MG1655 was obtained. The Δhns mutant lysogen that was generated after Stx phage lysogenic infection had a repressed growth status and showed subdued group behavior, including biofilm formation and swarming motility, in comparison to the wild-type strain. The de-repression effect of the H-NS mutation on CRISPR-Cas activity was then verified. The results showed that cas gene expression was upregulated and the transformation efficiency of the wild-type CRISPR plasmids was decreased, which may indicate activation of the CRISPR-Cas system. Furthermore, the function of CRISPR-Cas on Stx2 phage lysogen was investigated by activating the CRISPR-Cas system, which contains an insertion of the protospacer regions of the Stx2 phage Min27. The phage release and toxin production of four lysogens harboring the engineered CRISPRs were investigated. Notably, in the supernatant of the Δhns mutant lysogen harboring the Min27 spacer, both the progeny phage release and the toxin production were inhibited after mitomycin C induction. These observations demonstrate that the H-NS mutation-activated CRISPR-Cas system plays a role in modifying the effects of the Stx2 phage lysogen. Our findings indicated that H-NS mutation-mediated CRISPR-Cas activation in E. coli protects bacteria against Stx2 phage lysogeny by inhibiting the phage release and toxin production of the lysogen.

Study of regulation of some key enzymes of L-alanine biosynthesis by Brevibacterium Flavum producer strains

The mechanisms of L-alanine overproduction by Brevibacterium flavum strain-producers were studied. It was shown that β-Cl-L-alanine is an inhibitor of some key enzymes involved in the synthesis of L-alanine: alanine transaminase and valine-pyruvate transaminase. Two highly active B. flavum GL1 and GL18 strain-producers, which are resistant to the inhibitory effect of β-Cl-L-alanine, were obtained using a parental B. flavum AA5 strain-producer, characterized by a reduced activity of alanine racemase (≥98%). It was demonstrated that the increased L-alanine synthesis efficiency observed in the strain-producer developed in this work is associated with the absence of inhibition of alanine transaminase by the end product of the biosynthesis reaction, as well as with the effect of derepression of both alanine transaminase and valine-pyruvate transaminase synthesis by the studied compound.

Изучение регуляции некоторых ключевых ферментов биосинтеза L-аланина у штаммов-продуцентовBrevibacterium flavum

The mechanisms of L-alanine overproduction by Brevibacterium flavum producer strains were studied. It was shown that beta-CI-L-alanine is an inhibitor of some key enzymes involved in the synthesis of L-alanine, including alanine transaminase and valine-pyruvate transaminase. Two highly active B. flavum GL1 and GL1 8 producer strains, which are resistant to the inhibitory effect of beta-Cl-L-alanine, were obtained using a parental B. flavum AA5 producer strain, characterized by a reduced activity of alanine racemase (>or=98%). It was demonstrated that the increased L-alanine synthesis efficiency observed in the producer strains developed in this work is associated with the absence of inhibition of alanine transaminase by the end product of the biosynthesis reaction, as well as with the effect of derepression of both alanine transaminase and valine-pyruvate transaminase synthesis by the studied compound.

Dot1 binding induces chromatin rearrangements by histone methylation-dependent and -independent mechanisms

BackgroundMethylation of histone H3 lysine 79 (H3K79) by Dot1 is highly conserved among species and has been associated with both gene repression and activation. To eliminate indirect effects and examine the direct consequences of Dot1 binding and H3K79 methylation, we investigated the effects of targeting Dot1 to different positions in the yeast genome.ResultsTargeting Dot1 did not activate transcription at a euchromatic locus. However, chromatin-bound Dot1 derepressed heterochromatin-mediated gene silencing over a considerable distance. Unexpectedly, Dot1-mediated derepression was established by both a H3K79 methylation-dependent and a methylation-independent mechanism; the latter required the histone acetyltransferase Gcn5. By monitoring the localization of a fluorescently tagged telomere in living cells, we found that the targeting of Dot1, but not its methylation activity, led to the release of a telomere from the repressive environment at the nuclear periphery. This probably contributes to the activity-independent derepression effect of Dot1.ConclusionsTargeting of Dot1 promoted gene expression by antagonizing gene repression through both histone methylation and chromatin relocalization. Our findings show that binding of Dot1 to chromatin can positively affect local gene expression by chromatin rearrangements over a considerable distance.

Transcriptional De-Repression and Mfd Are Mutagenic in Stressed Bacillus subtilis Cells

In recent years, it has been proposed that conflicts between transcription and active chromosomal replication engender genome instability events. Furthermore, transcription elongation factors have been reported to prevent conflicts between transcription and replication and avoid genome instability. Here, we examined transcriptional de-repression as a genetic diversity-producing agent and showed, through the use of physiological and genetic means, that transcriptional de-represssion of a leuC defective allele leads to accumulation of Leu⁺ mutations. We also showed, by using riboswitches that activate transcription in conditions of tyrosine or methionine starvation, that the effect of transcriptional de-repression of the leuC construct on the accumulation of Leu⁺ mutations was independent of selection. We examined the role of Mfd, a transcription elongation factor involved in DNA repair, in this process and showed that proficiency of this factor promotes mutagenic events. These results are in stark contrast to previous reports in Escherichia coli, which showed that Mfd prevents replication fork collapses. Because our assays place cells under non-growing conditions, by starving them for two amino acids, we surmised that the Mfd mutagenic process associated with transcriptional de-repression does not result from conflicts with chromosomal replication. These results raise the interesting concept that transcription elongation factors may serve two functions in cells. In growing conditions, these factors prevent the generation of mutations, while in stress or non-growing conditions they mediate the production of genetic diversity.

Bmi-1 Is Dispensable for the Development of Acute Myeloid Leukemia Mediated by MLL-AF9

AbstractAbstract 63The Polycomb group 1 gene Bmi1 has been shown to be required for several normal adult stem cell types, including hematopoietic stem cells. High expression of Bmi1 is correlated with adverse prognosis in human AML and MDS. Bmi1 null murine fetal liver cells transformed with HoxA9 and Meis1 give rise to a primary leukemia of expanding blasts that fails to expand in secondary recipient mice thus demonstrating a defect in leukemia self-renewal (Nature. 2003; 423:255). More recently, Bmi1 has also been shown to bind PML-RARA, and to be required for in vitro replating of PML-RARA immortalized murine colony forming cells (cfc).Our laboratory recently demonstrated that MLL-AF9, a recurrent translocation commonly found in human acute leukemia, more efficiently transforms murine granulocyte macrophage progenitors (GMP) compared to the combination of HoxA9 and Meis1. We decided to test if MLL-AF9 is able to bypass the requirement for Bmi1 previously reported in other murine leukemia models. Lineage marker negative (Lin-) bone marrow cells from WT and Bmi1 −/− mice were transduced with an MSCV-based ecotropic retroviral vector expressing MLL-AF9 and linked via an internal ribosomal entry site (IRES) the selectable marker, GFP. Cells were harvested and injected into 5 sublethally (200 cGy) irradiated NOD/SCID mice per group. All mice developed AML based on GFP expression, spleen size, forward/side scatter profile and surface marker staining (Mac1 positive/Gr1 positive, B220 negative, CD3 negative). Median latency in the WT group was 54 days, whereas mice receiving a graft consisting of Bmi1 −/− cells transduced with MLL-AF9 developed leukemia with a median latency of 89 days. The experiment was repeated with very similar results with median latencies of 50 days (WT group) and 70 days (Bmi1 −/−). To confirm sustained self-renewal, we then proceeded to secondary transplantation experiments. Secondary leukemia developed with similar latency in recipients of primary WT (31.5d) and Bmi1−/− (32d) leukemic bone marrow cells. The experiment was repeated with bone marrow from mice with primary leukemias from the second cohort and again similar latencies were observed (42.5 days in the WT group vs 29.5 days in the Bmi1 −/− group.). Finally we proceeded to tertiary transplantation experiments. All recipients of secondary AML cells developed AML with median latencies of 17.5 d (WT) and 21 d (KO).To compare our results with a system closer to the original studies of leukemogenesis in a Bmi1 −/− background, we chose to transduce lin- bone marrow from WT and Bmi1 −/− with retroviral vectors for HoxA9-IRES-GFP and Meis1-pgk-puro. Both, WT and HoxA9/Meis1 transduced cells replated in in vitro methylcellulose cultures for at least 4 rounds. In the first of 2 in vivo experiments, 5/5 recipients of wild type cells developed acute myeloid leukemia with a median latency of 88 days. In contrast, none of the five recipients of HoxA9/Meis1a transduced Bmi1−/− cells developed leukemia, and these mice were sacrificed 6 months after transplantation. In a second independent experiment, 5/5 recipients of WT HoxA9/Meis1 transduced cells developed AML with a median latency of 62 days. One recipient of Bmi1 −/− cells transduced with HoxA9/Meis1 developed leukemia. One mouse died from a spontaneous lymphoma. The remaining 3 mice in this cohort did not develop leukemia. These findings are consistent with the previously published data and suggest that MLL-AF9 is more efficient in generating AML in Bmi1−/− bone marrow compared to the combination of HoxA9 and Meis1.To further characterize the Bmi1 −/− leukemias, we analyzed expression of the primary Bmi1 target p16 by Western blot. In primary, secondary and tertiary AMLs from Bmi1 −/− mice, derepression of p16 could be demonstrated. We analyzed the cell cycle distribution of cultured tertiary leukemia cells. WT cells showed a S phase proportion of 44–45 percent (n=2), whereas the Bmi1 −/− cells demonstrated a S-phase proportion of 30–38% (n=2). These results demonstrate modest effects of p16 derepression on cell cycle without catastrophic failure to divide. This is mirrored by the in vivo development of secondary and tertiary leukemias in the presence of elevated p16 levels. Preliminary data suggest a similar but less pronounced derepression of p19. Future experiments will focus on the mechanisms that allow MLL-AF9 transduced cells to grow in the presence of derepressed p16 and p19. Disclosures:Armstrong:Epizyme, Inc: Consultancy.

Derepressive effect of NH on hydrogen production by deleting the glnA1 gene in Rhodobacter sphaeroides

Purple non-sulfur (PNS) bacteria produce hydrogen by photofermentation of organic acids in wastewater. However, NH(4)(+) in wastewater may inhibit hydrogen synthesis by repressing the expression and activity of nitrogenase, the enzyme catalyzing hydrogen production in PNS bacteria. In this study, the Rhodobacter sphaeroides 6016 glnA gene encoding glutamine synthetase (GS) was knocked out by homologous recombination, and the effects on hydrogen production and nitrogenase activity were examined. Using 3 mM glutamine as the nitrogen source, hydrogen production (1,245-1,588 mL hydrogen/L culture) and nitrogenase activity were detected in the mutant in the presence of relatively high NH(4)(+) concentrations (15-40 mM), whereas neither was detected in the wild-type strain under the same conditions. Further analysis indicated that high NH(4)(+) concentrations greatly inhibited the expression of nifA and nitrogenase gene in the wild-type strain but not in the glnA1(-) mutant. These observations suggest that GS is essential to NH(4)(+) repression of nitrogenase and that deletion of glnA1 results in the complete derepression of nitrogenase by preventing NH(4)(+) assimilation in vivo, thus relieving the inhibition of nifA and nitrogenase gene expression. Knocking out glnA1 therefore provides an efficient approach to removing the inhibitory effects of ammonium ions in R. sphaeroides and possibly in other hydrogen-producing PNS bacteria.

The orphan nuclear receptor Rev-erbβ recruits Tip60 and HDAC1 to regulate apolipoprotein CIII promoter

Nuclear hormone receptors function as ligand activated transcription factors. Ligand binding and modification such as acetylation have been reported to regulate nuclear hormone receptors. The orphan receptors, Rev-erbα and Rev-erbβ, are members of the nuclear receptor superfamily and act as transcriptional repressors. In this study, the role of recruitment of co-factors by Rev-erbβ and acetylation of Rev-erbβ in modulating apolipoprotein CIII (apoCIII) transcription were investigated. Rev-erbβ was found to transcriptionally repress apoCIII after binding to the apoCIII promoter. Tip60, a histone acetyl-transferase (HAT), was a novel binding partner for Rev-erbβ and recruited to the apoCIII promoter by Rev-erbβ. Tip60 was able to acetylate Rev-erbβ and relieve the apoCIII repression mediated by Rev-erbβ. This de-repression effect depended on acetylation of Rev-erbβ at its RXKK motif by Tip60. In addition, histone deacetylase 1 (HDAC1) interacted with Rev-erbβ and was recruited to the apoCIII promoter by Rev-erbβ to antagonize Tip60's activity. Taken together, we have provided evidence that Rev-erbβ modulates the apoCIII gene expression by recruiting different transcription co-activator or co-repressor.

Light-dependent regulation of the gene cut-1 of Neurospora, involved in the osmotic stress response

The gene cut-1 from Neurospora encodes a protein of the haloacid dehalogenase (HAD) family, a group of enzymes usually associated to phosphatase or phosphotransferase activities. Loss of cut-1 function results in an osmosensitive phenotype and its transcription is induced by high osmotic pressure or heat-shock, but not by other stressing conditions tested. Unexpectedly, cut-1 transcript levels markedly decrease following illumination in the wild type and in some mutants tested, such as bd and vvd. The reduction was less pronounced in wc-1 and wc-2 mutants, indicating that this photoresponse is principally mediated by the WC system, a conclusion further supported by the lack of effect of blue light in a wc-1 mutant. Additionally, cut-1 mRNA levels are much higher in the dark in wc, vvd, bd, and cut mutants, showing the involvement of these genes in a cut-1 down-regulation mechanism. In the cases investigated, the mRNA levels of these strains increased further under high osmotic conditions, indicating an independent derepression effect. The environmental or genetic factors influencing cut-1 expression have no significant effect on the mRNA levels of two additional Neurospora genes encoding HAD proteins.

Free Fatty Acids Repress the GLUT4 Gene Expression in Cardiac Muscle via Novel Response Elements

Hyperlipidemia (HL) impairs cardiac glucose homeostasis, but the molecular mechanisms involved are yet unclear. We examined HL-regulated GLUT4 and peroxisome proliferator-activated receptor (PPAR) gamma gene expression in human cardiac muscle. Compared with control patients, GLUT4 protein levels were 30% lower in human cardiac muscle biopsies from patients with HL and/or type 2 diabetes mellitus, whereas GLUT4 mRNA levels were unchanged. PPARgamma mRNA levels were 30-50% lower in patients with HL and/or diabetes mellitus type 2 than in controls. Reporter studies in H9C2 cardiomyotubes showed that HL in vitro, induced by high levels of arachidonic (AA) stearic, linoleic, and oleic acids (24 h, 200 mum) repressed transcription from the GLUT4 promoter; AA also repressed transcription from the PPARgamma1 and PPARgamma2 promoters. Co-expression of PPARgamma2 repressed GLUT4 promoter activity, and the addition of AA further enhanced this effect. 5'-Deletion analysis revealed three GLUT4 promoter regions that accounted for AA-mediated effects: two repression-mediating sequences at -443/-423 bp and -222/-197 bp, the deletion of either or both of which led to a partial derepression of promoter activity, and a third derepression-mediating sequence at -612/-587 bp that was required for sustaining this derepression effect. Electromobility shift assay further shows that AA enhanced binding to two of the three regions of cardiac nuclear protein(s), the nature of which is still unknown. We propose that HL, exhibited as a high free fatty acid level, modulates GLUT4 gene expression in cardiac muscle via a complex mechanism that includes: (a) binding of AA mediator proteins to three newly identified response elements on the GLUT4 promoter gene and (b) repression of GLUT4 and the PPARgamma genes by AA.

Estrogen down-regulation of the corepressor N-CoR: Mechanism and implications for estrogen derepression of N-CoR-regulated genes

The nuclear receptor corepressor N-CoR plays a crucial role in the repressive activity of diverse transcription factors, yet little is known about what regulates its cellular level. We have found that estrogen markedly down-regulates N-CoR protein levels in estrogen receptor (ER)-positive breast cancer cells without affecting N-CoR mRNA levels, whereas levels of the related corepressor SMRT are unaffected. This effect is attributable to estrogen up-regulation of the ubiquitin ligase Siah2, which is a rapid and primary transcriptional response mediated by the ER, and precedes the loss of N-CoR. Treatment with proteasomal inhibitor or with small interfering RNA against Siah2 prevented the down-regulation of N-CoR by estrogen. Furthermore, the expression of 24-hydroxylase, a gene repressed by unliganded vitamin D receptor through its interaction with N-CoR, was up-regulated by estrogen and required Siah2. Our results illustrate a mechanism by which the estrogen-ER complex markedly reduces the level of N-CoR through a process involving the up-regulation of Siah2 and the subsequent targeting of N-CoR for proteasomal degradation. These findings reveal that, although estrogen directly regulates the transcription of many genes, by regulating a gene such as Siah2 it can exert profound "secondary" effects on cellular activity through mechanisms such as targeting regulatory proteins for degradation. This estrogen-evoked down-regulation of N-CoR could have a global derepressive effect on genes whose repression depends on N-CoR and thereby have broad impact on the activity of transcription factors and nuclear receptors whose actions involve N-CoR.

Lithium Can Relieve Translational Repression of TOP mRNAs Elicited by Various Blocks along the Cell Cycle in a Glycogen Synthase Kinase-3- and S6-Kinase-independent Manner

TOP mRNAs are translationally controlled by mitogenic, growth, and nutritional stimuli through a 5'-terminal oligopyrimidine tract. Here we show that LiCl can alleviate the translational repression of these mRNAs when progression through the cell cycle is blocked at G(0), G(1)/S, or G(2)/M phases in different cell lines and by various physiological and chemical means. This derepressive effect of LiCl does not involve resumption of cell division. Unlike its efficient derepressive effect in mitotically arrested cells, LiCl alleviates inefficiently the repression of TOP mRNAs in amino acid-deprived cells and has no effect in lymphoblastoids whose TOP mRNAs are constitutively repressed even when they are proliferating. LiCl is widely used as a relatively selective inhibitor of glycogen synthase kinase-3. However, inhibition per se of this enzyme by more specific drugs failed to derepress the translation of TOP mRNAs, implying that relief of the translational repression of TOP mRNAs by LiCl is carried out in a glycogen synthase kinase-3-independent manner. Moreover, this effect is apparent, at least in some cell lines, in the absence of S6-kinase 1 activation and ribosomal protein S6 phosphorylation, thus further supporting the notion that translational control of TOP mRNAs does not rely on either of these variables.

Antisense silencing of the creA gene in Aspergillus nidulans.

Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was approximately one-half of that achieved in a null creA mutant. Unlike results for that mutant, however, growth parameters and colony morphology in the antisense transformants were not affected.

Translational Control of Cyclin B1 mRNA during Meiotic Maturation: Coordinated Repression and Cytoplasmic Polyadenylation

Translational control is prominent during meiotic maturation and early development. In this report, we investigate a mode of translational repression in Xenopus laevis oocytes, focusing on the mRNA encoding cyclin B1. Translation of cyclin B1 mRNA is relatively inactive in the oocyte and increases dramatically during meiotic maturation. We show, by injection of synthetic mRNAs, that the cis-acting sequences responsible for repression of cyclin B1 mRNA reside within its 3′UTR. Repression can be saturated by increasing the concentration of reporter mRNA injected, suggesting that the cyclin B1 3′UTR sequences provide a binding site for a trans-acting repressor. The sequences that direct repression overlap and include cytoplasmic polyadenylation elements (CPEs), sequences known to promote cytoplasmic polyadenylation. However, the presence of a CPE per se appears insufficient to cause repression, as other mRNAs that contain CPEs are not translationally repressed. We demonstrate that relief of repression and cytoplasmic polyadenylation are intimately linked. Repressing elements do not override the stimulatory effect of a long poly(A) tail, and polyadenylation of cyclin B1 mRNA is required for its translational recruitment. Our results suggest that translational recruitment of endogenous cyclin B1 mRNA is a collaborative effect of derepression and poly(A) addition. We discuss several molecular mechanisms that might underlie this collaboration.

A strong carbon source effect is mediated by pUC plasmid sequences in a series of classical yeast vectors designed for promoter characterization.

While using YIp356 and YEp356R lacZ reporter plasmids, we found lacZ expression driven by the ARG2 promoter to be much higher in cells grown on a non-glucose carbon source than in glucose-grown cells (5-10-fold higher on galactose and up to 40-fold higher on ethanol). Furthermore, expression increased 30-fold upon shifting from a high-glucose to a low-glucose medium. This carbon source regulation requires Snf1p and possibly Ssn6p. It appears, however, to be artefactually mediated by plasmid sequences located upstream from the multicloning site. This emerged from the following observations: (a) the derepressive effect disappears if any extra piece of DNA is inserted upstream from the ARG2 promoter; and (b) similar derepression on low glucose is observed with another yeast promoter (ARG11), provided that the flanking 5' region is short. We determined that the cis-elements responsible for this physiologically irrelevant glucose regulation are located between positions 636 and 879 of the pUC18 DNA sequence.

Interactions of gender, growth hormone, and phenobarbital induction on murine Cyp2b expression

The interactions of gender, growth hormone, and phenobarbital induction on Cyp2b expression were examined in phenotypically normal ( lit/+) and growth-hormone deficient “little” ( lit/lit) mice. Using an immunocrossreactive monoclonal antibody designed to identify rat CYP2B1 and 2B2 proteins, we observed three hepatic Cyp2b proteins in control ( lit/+) females, but only two proteins, one at trace levels, in control males. Phenobarbital administration to lit/+ mice increased the expression of the two Cyp2b isoforms in the males by 3- to 4-fold, but produced an approximately 75% reduction in the female-expressed proteins. Whereas growth hormone depletion ( lit/lit) had no effect on the expression profile of Cyp2b proteins in females, it had a de-repressive effect in males, resulting in the expression of three proteins at concentrations now comparable to those observed in female liver. Generally, phenobarbital had no inductive effects in the lit/lit mice of both sexes. In all groups, transcript levels measured by a CYP2B1 probe were in agreement with the protein findings. In contrast, Cyp2b mRNA identified by an oligonucleotide probe for CYP2B2 were repressed completely by growth hormone in both sexes, and was expressed as a female-predominant transcript in the lit/lit mice. In spite of an apparent high degree of sequence homology between the rat CYP2B and murine Cyp2b gene families, the present findings highlight fundamental differences in their constitutive and gender-dependent expression, growth hormone regulation, and phenobarbital inducibility.