Nrg1 functions as a global transcriptional repressor of glucose-repressed genes through its direct binding to the specific promoter regions

Nrg1 is a zinc finger protein involved in the glucose repression of several glucose-repressed genes such as STA1, SUC2, and GAL1. Although the molecular details of the Nrg1-mediated repression of STA1 have been partly characterized, it still remains largely unknown how Nrg1 regulates these multiple target genes. In this study, we show that Nrg1 mediates the glucose repression of SUC2 and HXT2 through its direct binding to the specific promoter regions; it binds to the −404 to −360 region of the SUC2 promoter and the −957 to −810 region of the HXT2 promoter. Nrg1 also interacts with the −380 to −250 region of the PCK1 promoter, suggesting that it might also contribute to the PCK1 repression. In addition, ChIP assays confirmed that Nrg1 associated with specific promoter regions of these glucose-repressed genes in vivo. Analysis of the DNA fragments to which it binds indicates that Nrg1 may recognize T/ACCCC sequence within the promoters of these glucose-repressed genes as well as in its own promoter. Collectively, our findings indicate that Nrg1 mediates the glucose repression of multiple genes through its direct binding to the specific promoter regions.     

The expression of the rice (Oryza sativa L.) homologue of Snm1 is induced by DNA damages

We isolated and characterized the rice homologue of the DNA repair gene Snm1 (OsSnm1). The length of the cDNA was 1862bp; the open reading frame encoded a predicted product of 485 amino acid residues with a molecular mass of 53.2kDa. The OsSnm1 protein contained the conserved beta-lactamase domain in its internal region. OsSnm1 was expressed in all rice organs. The expression was induced by MMS, H(2)O(2), and mitomycin C, but not by UV. Transient expression of an OsSnm1/GFP fusion protein in onion epidermal cells revealed the localization of OsSnm1 to the nucleus. These results suggest that OsSnm1 is involved not only in the repair of DNA interstrand crosslinks, but also in various other DNA repair pathways.     

Development of anaerobically inducible nar promoter expression vectors for the expression of recombinant proteins in Escherichia coli

Dissolved oxygen (DO)-controlled nar promoter expression vectors were constructed, and their expression efficiency was compared with that of the T7 promoter pET22 expression vector by expressing human growth hormone (hGH), enhanced green fluorescence protein (EGFP), and β-tyrosinase in Escherichia coli cells. The nar promoter expression vector pRBS, which was engineered with a 5′-untranslated region and ribosomal binding site for the T7 promoter, expressed hGH at a rate of up to 32% of the total cellular proteins (TCP) in E. coli W3110narL⁻. The expression level of hGH was further enhanced, up to ∼42% of the TCP, by adding the N-terminal peptide tag of β-galactosidase to hGH, which was comparable to the expression of ∼43% of the TCP in pET-lac:hGH/BL21(DE3). A further engineered expression vector, pRBS(fnr), which coexpressed fumarate/nitrate reductase (fnr), expressed more EGFP than pET22 in BL21(DE3). In addition, recombinant β-tyrosinase was successfully expressed at a rate of up to ∼45% of the TCP in pRBS(fnr) in W3110narL⁻. From these results, the DO-controlled nar promoter system developed in this study can be considered a reliable and cost-effective expression system for protein production, especially in large-scale fermentation, as an alternative to the pET/BL(DE3) system.     

Coordinate regulation of bovine prion protein gene promoter activity by two Sp1 binding site polymorphisms

Relationships between insertion/deletion (Ins/Del) polymorphisms of the bovine prion protein gene (PRNP) promoter and bovine spongiform encephalopathy (BSE) susceptibility have been reported. Our previous study has shown that polymorphisms of −6C → T included in the specific protein 1 (Sp1) site in the 5′-flanking region of bovine PRNP influence the promoter activity of bovine PRNP. The present study shows that 12 and 23 bp Ins/Del polymorphisms in the upstream region and an additional polymorphism (−47C → A) in the Sp1 binding site coordinately affect the promoter activity. Reporter gene assays demonstrated that the bovine PRNP promoter containing −47A and 23 bp Del/12 bp Ins or 23 bp Ins/12 bp Ins showed lower promoter activity compared with other haplotypes (23 bp Del/12 bp Ins or 23 bp Ins/12 bp Del with −47C) or the wild-type haplotype (23 bp Del/12 bp Del with −47C). Furthermore, gel shift assays showed that the binding activity of Sp1 to the PRNP promoter was influenced by both polymorphisms with corresponding effects on the promoter activity. The coordinate regulation of the bovine PRNP promoter suggests the two Sp1 binding site polymorphisms control Sp1 binding to the PRNP promoter and its activity.     

AGO1 controls arabidopsis inflorescence architecture possibly by regulating TFL1 expression

Background and Aims

The TERMINAL FLOWER 1 (TFL1) gene is pivotal in the control of inflorescence architecture in arabidopsis. Thus, tfl1 mutants flower early and have a very short inflorescence phase, while TFL1-overexpressing plants have extended vegetative and inflorescence phases, producing many coflorescences. TFL1 is expressed in the shoot meristems, never in the flowers. In the inflorescence apex, TFL1 keeps the floral genes LEAFY (LFY) and APETALA1 (AP1) restricted to the flower, while LFY and AP1 restrict TFL1 to the inflorescence meristem. In spite of the central role of TFL1 in inflorescence architecture, regulation of its expression is poorly understood. This study aims to expand the understanding of inflorescence development by identifying and studying novel TFL1 regulators.

Methods

Mutagenesis of an Arabidopsis thaliana line carrying a TFL1::GUS (β-glucuronidase) reporter construct was used to isolate a mutant with altered TFL1 expression. The mutated gene was identified by positional cloning. Expression of TFL1 and TFL1::GUS was analysed by real-time PCR and histochemical GUS detection. Double-mutant analysis was used to assess the contribution of TFL1 to the inflorescence mutant phenotype.

Key Results

A mutant with both an increased number of coflorescences and high and ectopic TFL1 expression was isolated. Cloning of the mutated gene showed that both phenotypes were caused by a mutation in the ARGONAUTE1 (AGO1) gene, which encodes a key component of the RNA silencing machinery. Analysis of another ago1 allele indicated that the proliferation of coflorescences and ectopic TFL1 expression phenotypes are not allele specific. The increased number of coflorescences is suppressed in ago1 tfl1 double mutants.

Conclusions

The results identify AGO1 as a repressor of TFL1 expression. Moreover, they reveal a novel role for AGO1 in inflorescence development, controlling the production of coflorescences. AGO1 seems to play this role through regulating TFL1 expression.     

Cloning and expression analysis of a Parkinson's disease gene, uch-L1, and its promoter in zebrafish

Three genes, alpha-synuclein, parkin, and ubiquitin C-terminal hydrolase L1 (UCH-L1), have been associated with inherited forms of Parkinson's disease (PD), although their in vivo functions have remained largely unknown. To develop an animal model for the molecular study of PD, we cloned zebrafish uch-L1 cDNA and its gene promoter. Sequence analysis revealed that the zebrafish Uch-L1 is highly homologous (79%) to the human UCH-L1, which is a member of the deubiquitinating enzymes. By whole-mount in situ hybridization, we examined the spatiotemporal expression of uch-L1 mRNA in developing zebrafish embryos. The uch-L1 mRNAs are detected in neuronal cells at the first day of embryo development. The expression domain of uch-L1 overlaps with that of tyrosine hydroxylase, a molecular marker for dopaminergic neurons, in the ventral diencephalon, an equivalent structure to the substantia nigra where PD progresses in human. To further analyze the tissue-specific regulation of uch-L1 gene expression, we also tested its gene promoter activity and showed a preferential neuronal expression in transient transgenic zebrafish embryos. These results suggest that uch-L1 may have an important role in the development of neuronal cells in early embryos as well as in the degeneration and disease of neuronal cells in late adult brain.     

The rice flattened shoot meristem, encoding CAF-1 p150 subunit, is required for meristem maintenance by regulating the cell-cycle period

We isolated flattened shoot meristem (fsm) mutants in rice that showed defective seedling growth and died in the vegetative phase. Since most fsm plants had flat and small shoot apical meristems (SAMs), we suggest that FSM is required for proper SAM maintenance. FSM encodes a putative ortholog of Arabidopsis FASCIATA1 (FAS1) that corresponds to the p150 subunit of chromatin assembly factor-1 (CAF-1). FSM is expressed patchily in tissues with actively dividing cells, suggesting a tight association of FSM with specific cell-cycle phases. Double-target in situ hybridization counterstained with cell-cycle marker genes revealed that FSM is expressed mainly in the G₁ phase. In fsm, expressions of the two marker genes representing S- and G₂- to M-phases were enhanced in SAM, despite a reduced number of cells in SAM, suggesting that S- and G₂-phases are prolonged in fsm. In addition, developmental events in fsm leaves took place at the proper time, indicating that the temporal regulation of development occurs independently of the cell-cycle period. In contrast to the fasciated phenotype of Arabidopsis fas1, fsm showed size reduction of SAM. The opposite phenotypes between fsm and fas1 indicate that the SAM maintenance is regulated differently between rice and Arabidopsis.     

Naegleria fowleri: functional expression of the Nfa1 protein in transfected Naegleria gruberi by promoter modification

To establish a transient transfection system in a Naegleria, we constructed three nfa1-pEGFP-N1 vectors by the promoter replacement and insertion of a nfa1 gene and transfected the DNAs into Naegleria gruberi using a lipid reagent. The transfection efficiency and usefulness of the three modified vectors were estimated by identifying the expressions of the EGFP and Nfa1 protein from N. gruberi. After transfection, the Nfa1 protein was functionally expressed on pseudopodia of N. gruberi. The strong GFP fluorescence was observed in N. gruberi transfected with the actin-nfa1-pEGFP-N1 vector, of which the CMV promoter region in the expression vector was replaced with the actin 5' UTR region. Additionally, when transgenic N. gruberi trophozoites were co-cultured with CHO target cells, the Nfa1 protein was also located on cytoplasm and pseudopodia, especially on a food cup that was formed in contact with target cells as it shown in pathogenic N. fowleri.