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BBA - Gene Structure and Expression (v.1769, #7-8)
A facelift for the general transcription factor TFIIA by Torill Høiby; Huiqing Zhou; Dimitra J. Mitsiou; Hendrik G. Stunnenberg (pp. 429-436).
TFIIA was classified as a general transcription factor when it was first identified. Since then it has been debated to what extent it can actually be regarded as “general”. The most notable feature of TFIIA is the proteolytical cleavage of the TFIIAαβ into a TFIIAα and TFIIAβ moiety which has long remained a mystery. Recent studies have showed that TFIIA is cleaved by Taspase1 which was initially identified as the protease for the proto-oncogene MLL. Cleavage of TFIIA does not appear to serve as a step required for its activation as the uncleaved TFIIA in the Taspase1 knock-outs adequately support bulk transcription. Instead, cleavage of TFIIA seems to affect its turn-over and may be a part of an intricate degradation mechanism that allows fine-tuning of cellular levels of TFIIA. Cleavage might also be responsible for switching transcription program as the uncleaved and cleaved TFIIA might have distinct promoter specificity during development and differentiation. This review will focus on functional characteristics of TFIIA and discuss novel insights in the role of this elusive transcription factor.
Keywords: TFIIA; Transcription; Taspase1; Cleavage
Expression of HOXC9 and E2F2 are up-regulated in CD133+ cells isolated from human astrocytomas and associate with transformation of human astrocytes by Oswaldo K. Okamoto; Sueli M. Oba-Shinjo; Luciana Lopes; Suely K. Nagahashi Marie (pp. 437-442).
Comparative analysis of cancer stem cells with their neoplastic and non-neoplastic counterparts should help better understand the underlying molecular events leading to transformation and tumor dissemination. Here, we report a molecular signature comprised by genes with exclusive aberrant expression in CD133+ cells, a reported subpopulation of tumorigenic stem-like cells, isolated from human glioblastomas. Microarrays covering 55,000 transcripts were used to compare gene expression profiles in purified subpopulations of CD133+ and CD133− GBM cells. Sixteen genes, many of which not previously associated with astrocytomas, were found aberrantly expressed in CD133+ cells, but not in CD133−, when compared with corresponding non-neoplastic controls. Up-regulation of two of such genes, E2F2 and HOXC9, was detected in a set of 54 astrocytomas of different grades and significantly associated with malignancy. Due to their distinctive expression in CD133+ cells, the use of E2F2 and HOXC9 as therapeutic targets for tumor eradication is suggested.
Keywords: Cancer stem cell; CD133; Glioblastoma; Astrocytoma; Malignancy; Microarray
Differential regulation of Ku gene expression in etiolated mung bean hypocotyls by auxins by Pei-Feng Liu; Wen-Chi Chang; Yung-Kai Wang; Suresh-Babu Munisamy; Shen-Hsing Hsu; Hwan-You Chang; Shu-Hsing Wu; Rong-Long Pan (pp. 443-454).
Plant Ku genes were identified very recently in Arabidopsis thaliana, and their roles in repair of double-stranded break DNA and maintenance of telomere integrity were scrutinized. In this study, the cDNAs encoding Ku70 ( VrKu70) and Ku80 ( VrKu80) were isolated from mung bean ( Vigna radiata L.) hypocotyls. Both genes were expressed widely among different tissues of mung bean with the highest levels in hypocotyls and leaves. The VrKu gene expression was stimulated by exogenous auxins in a concentration- and time-dependent manner. The stimulation could be abolished by auxin transport inhibitors, N-(1-naphthyl) phthalamic acid and 2,3,5-triiodobenzoic acid implicating that exogenous auxins triggered the effects following their uptake by the cells. Further analysis using specific inhibitors of auxin signaling showed that the stimulation of VrKu expression by 2,4-dichlorophenoxyacetic acid (2,4-D) was suppressed by intracellular Ca2+ chelators, calmodulin antagonists, and calcium/calmodulin dependent protein kinase inhibitors, suggesting the involvement of calmodulin in the signaling pathway. On the other hand, exogenous indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NAA) stimulated VrKu expression through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Altogether, it is thus proposed that 2,4-D and IAA (or NAA) regulate the expression of VrKu through two distinct pathways.
Keywords: Abbreviations; BAPTA; 1,2-bis(2-aminophenoxy)ethane-; N,N,N′,N′; -tetraacetic acid; C5; lavendustin C; DEB; DNA end-binding; DNA–PK; DNA-dependent protein kinase; DSB; double-strand break; EDTA; N,N,N′N′; -ethylenediamine tetraacetic acid; EGTA; ethylene glycol-bis(2-aminoethylether)-; N,N,N′N′; -tetraacetic acid; EMSA; electrophoretic mobility shift assay; H7; 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine; IAA; indole-3-acetic acid; NAA; α-naphthalene acetic acid; NHEJ; non-homologous end joining; NPA; N; -(1-naphthyl) phthalamic acid; PACOCF3; palmitoyl trifluoromethyl ketone; PMSF; phenylmethylsulfonyl fluoride; TFP; trifluoperazine; TIBA; 2,3,5-triiodobenzoic acid; TMB-8 HCl; 8-(; N,N; -diethylamino)-octyl-3,4,5-trimethoxybenzoate HCl; W7; N; -(-6-amino-hexyl)-5-chloro-1-naphthalenesulpho-namide; 2,4-D; 2,4-dichlorophenoxyacetic acidAuxin; DNA end-binding; DNA repair; DNA double-strand breaks; Gene regulation; Ku protein
A conserved role of SHORT VEGETATIVE PHASE ( SVP) in controlling flowering time of Brassica plants by Jeong Hwan Lee; Soo Hyun Park; Jong Seob Lee; Ji Hoon Ahn (pp. 455-461).
The control of flowering time in Brassica plants is an important approach for improving productivity, as vegetative tissues are not produced after the floral transition in Brassica plants. In order to determine the feasibility of modulating flowering time in Chinese cabbage plants, genes homologous to Arabidopsis SHORT VEGETATIVE PHASE ( AtSVP) were isolated from spring-type and fall-type cultivars of Chinese cabbage plants, and their functions were determined. Their deduced amino acid sequences were 91–93% identical with that of AtSVP. The expression of BcSVP was ubiquitously detected, and was unaffected by vernalization. Constitutive BcSVP expression induced late flowering with additional floral defects. This delayed flowering was attributed to the repression of FLOWERING LOCUS T ( FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 ( SOC1). BcSVP expression under the control of the AtSVP promoter also resulted in the complementation of the svp mutation in Arabidopsis. These results indicate that BcSVP is a functional equivalent of AtSVP and also suggest that BcSVP may prove useful for the genetic manipulation of flowering time in Brassica plants.
Keywords: Chinese cabbage; BcSVP; Vernalization; Floral repressor; Flowering time; Genetic manipulation
Modulation of 16S rRNA function by ribosomal protein S12 by Anton Vila-Sanjurjo; Ying Lu; Jamie L. Aragonez; Rebekah E. Starkweather; Manoj Sasikumar; Michael O'Connor (pp. 462-471).
Ribosomal protein S12 is a critical component of the decoding center of the 30S ribosomal subunit and is involved in both tRNA selection and the response to streptomycin. We have investigated the interplay between S12 and some of the surrounding 16S rRNA residues by examining the phenotypes of double-mutant ribosomes in strains of Escherichia coli carrying deletions in all chromosomal rrn operons and expressing total rRNA from a single plasmid-borne rrn operon. We show that the combination of S12 and otherwise benign mutations at positions C1409–G1491 in 16S rRNA severely compromises cell growth while the level and range of aminoglycoside resistances conferred by the G1491U/C substitutions is markedly increased by a mutant S12 protein. The G1491U/C mutations in addition confer resistance to the unrelated antibiotic, capreomycin. S12 also interacts with the 912 region of 16S rRNA. Genetic selection of suppressors of streptomycin dependence caused by mutations at proline 90 in S12 yielded a C912U substitution in 16S rRNA. The C912U mutation on its own confers resistance to streptomycin and restricts miscoding, properties that distinguish it from a majority of the previously described error-promoting ram mutants that also reverse streptomycin dependence.
Keywords: Ribosomal protein S12; Paromomycin; Streptomycin; Capreomycin; Translation
Human peroxiredoxin 5 gene organization, initial characterization of its promoter and identification of alternative forms of mRNA by Nhu Tiên Nguyên-nhu; Jehanne Berck; André Clippe; Elee Duconseille; Hanane Cherif; Christophe Boone; Valérie Van der Eecken; Alfred Bernard; Ingrid Banmeyer; Bernard Knoops (pp. 472-483).
Peroxiredoxin 5 (PRDX5) is a mammalian thioredoxin peroxidase ubiquitously expressed in tissues. Its role as antioxidant enzyme has been previously supported in different pathological situations. In this study, we determined the complete human PRDX5 genomic organization and isolated the 5′-flanking region of the gene. Human PRDX5 gene is composed of six exons and five introns similarly to other chordate PRDX5 genes. Several single nucleotide polymorphisms were identified. Six out of them have amino acid substitutions in protein-coding region. Analysis of the 5′-flanking region of human PRDX5 revealed the presence of a TATA-less promoter containing a canonical CpG island and several putative response elements for transcription factors. To analyze the regulatory mechanisms controlling human PRDX5 expression, we characterized the 5′-flanking region by cloning various segments of this region in front of a luciferase reporter sequence. Transfection in HepG2 cells indicate that the 5′-flanking region contains regulatory elements for constitutive expression of human PRDX5. Multiple transcription start sites were also identified by 5′-RACE-PCR in human liver. Moreover, although no corresponding proteins were reported, we present new alternative splicing variants encoded specifically by human PRDX5 gene. The characterization of human PRDX5 gene revealed the complexity of its regulation and a high variability of sequences that might be associated with pathological situations.
Keywords: Abbreviations; AP-1; Activator Protein-1; ARE; Antioxidant Response Element; GRE; Glucocorticoid Response Element; InRE; Insulin Response Element; NF-κB; Nuclear Factor κB; PRDX; peroxiredoxin; RACE; rapid amplification of cDNA ends; ROS; Reactive Oxygen Species; SNP; single nucleotide polymorphism; Sp1; Specific Protein-1Peroxiredoxin; Promoter; Transcription start site; Alternative splicing; Gene polymorphism
Flexible interaction of Drosophila Smad complexes with bipartite binding sites by Sheng Gao; Allen Laughon (pp. 484-496).
A subset of BMP-responsive enhancer elements are characterized by pairing of a GC-rich Smad1 binding site and an SBE-type Smad4 binding site. Such paired, or bipartite, sites are in some cases just 5 bp apart and thus might be contacted by a single Smad1–Smad4 complex. Other potential pairings are separated as much as 60 bp but it is not known whether such longer distances can be spanned by a Smad1–Smad4 complex, indeed binding of native Smad1–Smad4 complexes to any of these bipartite elements has yet to be reported. Here we report that a complex of the homologous Drosophila Smad proteins, Mad and Medea, is capable of concerted binding to GC-rich and SBE sites separated by as much as 20 bp. The wider the separation, the more severely binding affinity was reduced by shortening of the linker region that tethers the DNA binding domain of Medea. In contrast, length of the Mad linker did not affect the allowed distance between paired sites, rather it contributes specifically to Mad contact with the GC-rich site. Finally, we show that Smad1 and Smad4 can participate in binding to bipartite sites.
Keywords: Smad; Mad; Medea; Dpp; Brinker; BMP
Involvement of the elicitor-induced gene OsWRKY53 in the expression of defense-related genes in rice by Tetsuya Chujo; Ryota Takai; Chiharu Akimoto-Tomiyama; Sugihiro Ando; Eiichi Minami; Yoshiaki Nagamura; Hanae Kaku; Naoto Shibuya; Michiko Yasuda; Hideo Nakashita; Kenji Umemura; Atsushi Okada; Kazunori Okada; Hideaki Nojiri; Hisakazu Yamane (pp. 497-505).
We present a detailed characterization of the chitin oligosaccharide elicitor-induced gene OsWRKY53. OsWRKY53 was also induced in suspension-cultured rice cells by a fungal cerebroside elicitor and in rice plants by infection with the blast fungus Magnaporthe grisea. A fusion of OsWRKY53 with green fluorescent protein was detected exclusively in the nuclei of onion epidermal cells, and OsWRKY53 protein specifically bound to W-box elements. A transient assay using the particle bombardment method showed that OsWRKY53 is a transcriptional activator. A microarray analysis revealed that several defense-related genes, including pathogenesis-related protein genes such as PBZ1, were upregulated in rice cells overexpressing OsWRKY53. Finally, overexpression of OsWRKY53 in rice plants resulted in enhanced resistance to M. grisea. These results strongly suggest that OsWRKY53 is a transcription factor that plays important roles in elicitor-induced defense signaling pathways in rice.
Keywords: Defense response; Elicitor; Oryza sativa; L. cv. Nipponbare; WRKY
An enhancer-like region regulates hrp3 promoter stage-specific gene expression in the human malaria parasite Plasmodium falciparum by Carlos López-Estraño; Anusha M. Gopalakrishnan; Jean-Philippe Semblat; M. Ross Fergus; Dominique Mazier; Kasturi Haldar (pp. 506-513).
The asexual blood stage of Plasmodium falciparum is comprised of morphologically distinct ring, trophozoite and schizont stages. Each of these developmental stages possesses a distinct pattern of gene expression. Regulation of P. falciparum gene expression is thought to occur, at least in part, at the promoter level. Previously, we have found that although the hrp3 mRNA is only seen in ring-stage parasites, deletion of a specific sequence in the 5′ end of the promoter region decreased ring-stage expression of hrp3 and enabled detection of its transcripts in trophozoite-stage parasites. In order to investigate this stage specific regulation of gene expression, we employed a series of nested deletions of the 1.7-kb hrp3 promoter. Firefly luciferase gene was used as a reporter to evaluate the role of promoter sequences in gene regulation. Using this approach, we identified a ring-stage specific regulatory region on the hrp3 promoter located between −1.7 kb and −1.1 kb from the ATG initiation codon. Small 100–150 bp truncations on this enhancer-like region failed to uncover discrete regulatory sequences, suggesting the multipartite nature of this element. The data presented in this study demonstrate that stage specific promoter activity of the hrp3 gene in P. falciparum blood stage parasites is supported, at least in-part, by a small promoter region that can function in the absence of a larger chromosomal context.
Keywords: Abbreviations; dhfr-ts; dihydrofolate reductase-thymidylate synthase; bp; base pair; nt; nucleotides; cam; calmodulin; msp; 1; merozoite surface protein 1; hrp; 3; histidine-rich protein 3; hrp; 2; histidine-rich protein 2; PfEMP1; Plasmodium falciparum; erythrocyte membrane protein 1; hsp; 86; heat shock protein 86; pfs; 16; Plasmodium falciparum; sexual-stage antigen 16; pfs; 25; Plasmodium falciparum; sexual-stage antigen 25; RNA; ribonucleic acid; rRNA; ribosomal ribonucleic acid; CAT; chloramphenicol acetyl transferase; PCR; polymerase chain reaction; hpi; hours post-invasion; mRNA; messenger ribonucleic acid; rif; repetitively Interspersed Family; stevor; subtelomeric variable open reading frame; HDGFP; human dihydrofolate reductase fused to; Aequorea victoria; Green fluorescent protein Plasmodium; transcription; regulation; histidine-rich protein 3; stage specificity; expression
Cloning, genomic organization, and tissue-specific expression of the RASL11B gene by Katrin Stolle; Michael Schnoor; Georg Fuellen; Michael Spitzer; Paul Cullen; Stefan Lorkowski (pp. 514-524).
RASL11B is a member of the small GTPase protein family with a high degree of similarity to RAS proteins. Cloning of RASL11B mRNA and in silico analyses revealed that the human RASL11B gene spans about 4.5 kb and comprises four exons on chromosomal locus 4q12. The proximal 5′-flanking region of the gene lacks a TATA box but is GC-rich and contains a CCAAT box and several Sp1 sites. Consistent with this, the RASL11B gene was found to be expressed in all tissues investigated, with highest levels in placenta and in primary macrophages. The predicted RASL11B protein has no typical prenylation signal, indicating that it is probably not anchored to cellular membranes. RASL11B was induced during maturation of THP-1 monocytic cells into macrophage-like cells and in coronary artery smooth muscle cells after treatment with TGF-β1. These results indicate that RASL11B may play a role in TGF-β1-mediated developmental processes and in pathophysiologies such as inflammation, cancer, and arteriosclerosis.
Keywords: RASL11B; RASL11A; RAS-like protein; GTPase; Transforming growth factor β1; TGF-β1; Smooth muscle cell