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BBA - Gene Structure and Expression (v.1759, #11-12)
Regulation of Smad activities by Lan Xu (pp. 503-513).
TGF-β ( Transforming Growth Factor- β) cytokines employ Smad proteins as the intracellular mediator of signaling. Upon TGF-β stimulation, the cytoplasmic Smads become phosphorylated and consequently accumulate in the nucleus to regulate target gene expression. The cytoplasm-to-nucleus redistribution of Smads, as well as the ability of Smads to activate or repress gene transcription, is under multiple layers of regulation by factors not limited to TGF-β. With recent advance in the knowledge of regulatory factors impinged on Smads, we are beginning to understand the complexity in cellular responses to TGF-β.
Keywords: Smad; TGF-β; Nucleocytoplasmic trafficking; Phosphorylation; Ubiquitination; SUMOylation
Molecular cloning and characterization of human Aph2 gene, involved in AP-1 regulation by interaction with JAB1 by Fengrui Zhang; Yujun Di; Jinjun Li; Yan Shi; Liping Zhang; Chunyan Wang; Xianghuo He; Yongzhong Liu; Dafang Wan; Keke Huo; Jianren Gu (pp. 514-525).
A human Aph2 gene ( hAph2) was identified and cloned from a human placenta cDNA library. Bioinformatics analysis revealed hAPH2 protein shares 96% identity with mouse APH2 and contains a zf-DHHC domain (148–210aa), which is always involved in protein–protein or protein–DNA interaction. Differential expression patterns of hAph2 mRNA were observed in normal human tissues. Yeast two-hybrid screening found another hAPH2-interacting protein JAB1. The zf-DHHC domain of hAPH2 and the C-terminal of JAB1 were confirmed to be critical for the interaction. Fused with GFP and expressed in COS-7, NIH/3T3 and SMMC-7721 cell lines, hAPH2 showed predominant distribution in the cytoplasm and co-localized with JAB1 around the nucleus. Furthermore, overexpression of hAPH2 could increase apoptosis of COS-7 cells and negatively regulate JAB1-induced activation of AP-1 in a concentration dependent manner. The expression level of c-jun was also down-regulated by overexpression of hAPH2 in COS-7 cells. These data showed some basic characterization and function of hAph2 (hAPH2), dependent or independent with JAB1.
Keywords: hAph2; zf-DHHC; Yeast two-hybrid; JAB1; AP-1
Pf-ALMP, a novel astacin-like metalloproteinase with cysteine arrays, is abundant in hemocytes of pearl oyster Pinctada fucata by Xunhao Xiong; Lei Chen; You Li; Liping Xie; Rongqing Zhang (pp. 526-534).
The astacin family metalloproteinase is a family of zinc-dependent endopeptidases which play crucial roles in embryonic development, bone growth and morphogenesis. A cDNA clone encoding a putative astacin-like metalloproteinase (pf-ALMP) was isolated from hemocytes of pearl oyster, Pinctada fucata. The novel metalloproteinase presents a molecular organization close to the astacins, but has a novel C-terminal domain with cysteine arrays. RT-PCR analysis revealed that pf-ALMP was expressed dramatically high in hemocytes, which was affected by lipopolysaccharides (LPS) challenge. High expression of pf-ALMP was also found in gill, gonad and digestion gland, and in situ hybridization demonstrated that pf-ALMP was expressed in the epithelia cells of these tissues. Substrate analysis studies indicated that the recombinant pf-ALMP catalytic domain could digest gelatin. Interestingly, the pf-ALMP also could be involved in cell proliferation processes and the cysteine arrays were necessary for the proliferative activity. Taken together, these studies also help to further understand the functions of astacins which may be related to the processes of molluscan inflammatory response, embryo development, proliferation and shell formation.
Keywords: Astacin; Metalloproteinase; Hemocyte; Cysteine array; Extracellular matrix
mRNA made during heat shock enters the first round of translation by Laura Marín-Vinader; Siebe T. van Genesen; Nicolette H. Lubsen (pp. 535-542).
To determine whether mRNA synthesized during a heat shock is translated at least once in spite of the strong inhibition of translation by heat shock, we used nonsense-mediated decay (NMD) as an assay since NMD requires a round of translation. As NMD substrate we used the human ψγE-crystallin gene, which contains a premature termination codon, and as control, its close relative, the human γD-crystallin gene, both placed under control of the Hsp70 promoter. We show that no spliced ψγE-crystallin mRNA can be detected in heat shocked cells, suggesting that NMD resumes as soon as splicing is restored. We further show that newly synthesized mRNAs co-sediment with the 40S ribosomal subunits, indicating that the transcripts are recruited to the translation machinery but are stalled at the translation initiation stage. Using fluorescence loss in photobleaching (FLIP) we show that cytoplasmic EGFP-CBP20 is immobile in heat shocked cells. CBP20 is part of the cap binding complex which is thought to direct the first round of translation. Together our data suggest that all mRNAs made during heat shock enter the pioneer round of translation.
Keywords: Cap-binding complex; First round of translation; Stress; Splicing; CBP20
Identification and characterization of genes associated with the induction of embryogenic competence in leaf-protoplast-derived alfalfa cells by M. Domoki; J. Györgyey; J. Bíró; T.P. Pasternak; Á. Zvara; S. Bottka; L.G. Puskás; D. Dudits; A. Fehér (pp. 543-551).
Alfalfa leaf protoplast-derived cells can develop into somatic embryos depending on the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the initial culture medium. In order to reveal gene expression changes during the establishment of embryogenic competence, we compared the cell types developed in the presence of 1 and 10 μM 2,4-D, respectively, at the time of their first cell divisions (fourth day of culture) using a PCR-based cDNA subtraction approach. Although the subtraction efficiency was relatively low, applying an additional differential screening step allowed the identification of 38 10 μM 2,4-D up-regulated transcripts. The corresponding genes/proteins were annotated and representatives of various functional groups were selected for more detailed gene expression analysis. Real-time quantitative PCR (RT-QPCR) analysis was used to determine relative expression of the selected genes in 2,4-D-treated leaves as well as during the whole process of somatic embryogenesis. Gene expression patterns confirmed 2,4-D inducibility for all but one of the 11 investigated genes as well as for the positive control leafy cotyledon1 ( MsLEC1) gene. The characterized genes exhibited differential expression patterns during the early induction phase and the late embryo differentiation phase of somatic embryogenesis. Genes coding for a GST-transferase, a PR10 pathogenesis-related protein, a cell division-related ribosomal (S3a) protein, an ARF-type small GTPase and the nucleosome assembly factor family SET protein exhibited higher relative expression not only during the induction of somatic embryogenesis but at the time of somatic embryo differentiation as well. This may indicate that the expression of these genes is associated with developmental transitions (differentiation as well as de-differentiation) during the process of somatic embryogenesis.
Keywords: Somatic embryogenesis; 2,4-dichlorophenoxyacetic acid; Gene expression; Medicago sativa; L.; cDNA subtraction
Identification of hnRNPs K, L and A2/B1 as candidate proteins involved in the nutritional regulation of mRNA splicing by Brian N. Griffith; Callee M. Walsh; Wioletta Szeszel-Fedorowicz; Aaron T. Timperman; Lisa M. Salati (pp. 552-561).
Nutrient regulation of glucose-6-phosphate dehydrogenase (G6PD) expression occurs through changes in the rate of splicing of G6PD pre-mRNA. This posttranscriptional mechanism accounts for the 12- to 15-fold increase in G6PD expression in livers of mice that were starved and then refed a high-carbohydrate diet. Regulation of G6PD pre-mRNA splicing requires a cis-acting element in exon 12 of the pre-mRNA. Using RNA probes to exon 12 and nuclear extracts from livers of mice that were starved or refed, proteins of 60 kDa and 37 kDa were detected bound to nucleotides 65–79 of exon 12 and this binding was decreased by 50% with nuclear extracts from refed mice. The proteins were identified as hnRNPs K, L, and A2/B1 by LC-MS/MS. The decrease in binding of these proteins to exon 12 during refeeding was not accompanied by a decrease in the total amount of these proteins in total nuclear extract. HnRNPs K, L and A2/B1 have known roles in the regulation of mRNA splicing. The decrease in binding of these proteins during treatments that increase G6PD expression is consistent with a role for these proteins in the inhibition of G6PD mRNA splicing.
Keywords: RNA splicing; hnRNP; nutritional regulation; posttranscriptional gene regulation; lipogenesis; liver
Dusty protein kinases: Primary structure, gene evolution, tissue specific expression and unique features of the catalytic domain by Jianbin Peng; Wenji Dong; Ying Chen; Rong Mo; Jan-Fang Cheng; Chi-chung Hui; Narla Mohandas; Cheng-Han Huang (pp. 562-572).
Ser/Thr- and Tyr-Protein kinases constitute a key switch underlying the dynamic nature and graded regulation of signal transduction and pathway activities in cellular organization. Here we describe the identification and characterization of Dusty, a single-copy gene that arose in metazoan evolution and encodes a putative dual Ser/Thr and Tyr protein kinase with unique structural features. Dusty is widely expressed in vertebrates, broadly distributed in the central nervous system, and deregulated in certain human cancers. Confocal imaging of transiently expressed human Dusty-GFP fusion proteins showed a cytoplasmic distribution. Dusty proteins from lower to higher species display an increasing degree of sequence conservation from the N-terminal non-catalytic domain to C-terminal catalytic domain. The non-catalytic region has eight conserved cysteine residues, multiple potential kinase-docking motifs and phosphorylation sites, whereas the catalytic domain is divergent and about equally distant of Ser/Thr and Tyr protein kinases. Homology analyses identified the essential catalytic residues, suggesting that Dusty homologues all possess the enzymatic activity of a protein kinase. Taken together, Dusty is a unique evolutionarily selected group of divergent protein kinases that may play important functional roles in the brain and other tissues of vertebrates.
Keywords: Abbreviations; ePK; eukaryotic protein kinase catalytic domain; BAC; bacterial artificial chromosome; bp; basepair; EST; expressed sequence tags; FISH; fluorescence; in situ; hybridization; GFP; green fluorescence protein; GSP; gene-specific primer; kb; kilobase; nt; nucleotide; ORF; open reading frame; PCR; polymerase chain reaction; PSK; protein-serine/threonine kinase; PTK; protein-tyrosine kinase; RACE; rapid amplification of cDNA ends; RT-PCR; reverse transcriptase PCR; UTR; untranslated regionDusty protein kinase; Tissue specificity; Protein structure; Gene evolution; Vertebrate animal; Human cancer
The Sp family of transcription factors regulates the human laminin α1 gene in JAR choriocarcinoma cells by Tomoaki Niimi; Yoshitaka Hayashi; Kiyotoshi Sekiguchi; Yasuo Kitagawa (pp. 573-579).
Laminin-111 (α1β1γ1) is the major component of the embryonic and extra-embryonic basement membrane. The laminin α1 chain shows a restricted and developmentally regulated expression in basement membranes of distinct epithelial tissues while β1 and γ1 chains have a wide tissue distribution. To understand how human laminin α1 chain expression is controlled, we cloned and characterized the 5′-flanking region of the human laminin α1 ( LAMA1) gene. Transfection studies using serially deleted promoter constructs and JAR choriocarcinoma cells revealed that the minimal promoter fragment resided in the +31 to −206 region, which contains a number of GC- and GT/A-rich motifs for the binding of the Sp family of transcription factors. Electrophoretic mobility shift assays and mutational analyses revealed that Sp1 and Sp3 bound specifically to these elements and are important for the promoter activity. Furthermore, we showed that Krüppel-like factors KLF4 and KLF6 also activate transcription of the human LAMA1 gene. Chromatin immunoprecipitation analysis demonstrated recruitment of these transcription factors to the promoter region. These results indicate that transcription of the human LAMA1 gene is controlled by a combination of the actions of Sp1/Sp3 and Krüppel-like factors, KLF4 and KLF6.
Keywords: Laminin; Promoter; Sp family; Krüppel-like factors; JAR cell