Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
BBA - Gene Structure and Expression (v.1730, #3)
Gene selective suppression of nonsense termination using antisense agents by Agné Kulyté; Rikard Dryselius; Jenny Karlsson; Liam Good (pp. 165-172).
An estimated one third of all inherited genetic disorders and many forms of cancer are caused by premature (nonsense) termination codons. Aminoglycoside antibiotics are candidate drugs for a large number of such genetic diseases; however, aminoglycosides are toxic, lack specificity and show low efficacy in this application. Because translational termination is an active process, we considered that steric hindrance by antisense sequences could trigger the ribosome's “default mode� of readthrough when positioned near nonsense codons. To test this hypothesis, we performed experiments using plasmids containing a luciferase reporter with amber, ochre and opal nonsense mutations within the luxB gene in Escherichia coli. The nonspecific termination inhibitors gentamicin and paromomycin and six antisense peptide nucleic acids (PNA) spanning the termination region were tested for their potential to suppress the luxB mutation. Gentamicin and paromomycin increased luciferase activity up to 2.5- and 10-fold, respectively. Two of the PNAs increased Lux activity up to 2.5-fold over control levels, with no significant effect on cell growth or mRNA levels. Thus, it is possible to significantly suppress nonsense mutations within target genes using antisense PNAs. The mechanism of suppression likely involves enhanced readthrough, but this requires further investigation. Nonsense termination in human cells may also be susceptible to suppression by antisense agents, providing a new approach to address numerous diseases caused by nonsense mutations.
Keywords: Nonsense suppression; Readthrough; Antisense; Peptide nucleic acid; Translation termination
Analysis of mono-ADP-ribosyltransferase 4 gene expression in human monocytes: Splicing pattern and potential regulatory elements by Andreas Grahnert; Maik Friedrich; Kurt Engeland; Sunna Hauschildt (pp. 173-186).
Mono-ADP-ribosyltransferase (ART) 4 belongs to a family of ectoenzymes that catalyze the transfer of ADP-ribose from NAD+ to a target protein. ART4 could be detected on HEL cells and erythrocytes by FACS analysis while it was absent from activated monocytes, despite the presence of ART4 mRNA in these cells. The predicted glycosylphosphatidylinositol (GPI) linkage of ART4 could be verified by showing that treatment of erythrocytes, HEL cells and ART4-transfected HEK-293-T cells with phosphatidylinositol-specific phospholipase C results in a decrease in ART4 expression. Furthermore, an ART4 construct carrying an Ala285Val mutation that is critical for the formation of a GPI anchor failed to be expressed in transfected C-33A cells. Analysis of the gene structure revealed that the first of the three exons was at least 236 bp longer than previously published and that splicing occurred in the coding region of the mRNA from HEL cells and monocytes. When carrying out 5′ inverse RACE-PCR we confirmed the existence of 5 ATGs in the 5′ untranslated region (5′UTR). By deletion and site-directed mutagenesis of the ATGs, we showed that the first two ATGs impair translation and that both the 3rd and 5th ATG can be used for translation initiation after expression in C-33A cells. On analysis of the 3′UTR, which contains 2 adenylate/uridylate-rich elements (AREs), we detected one variant in monocytes that would be devoid of a GPI-anchor signal and thus could represent a secreted form of ART4. Thus, alternative splicing and the use of regulatory elements in the 5′UTR and 3′UTR represent means to control ART4 expression.
Keywords: Mono-ADP-ribosyltransferase; Inverse 5′ RACE-PCR; 5′UTR; 3′UTR; Monocyte; HEL cell
Molecular cloning of a molluscan gonadotropin-releasing hormone receptor orthologue specifically expressed in the gonad by Franck Rodet; Christophe Lelong; Marie-Pierre Dubos; Katherine Costil; Pascal Favrel (pp. 187-195).
Despite their economic importance, only very little information is available regarding (neuro)endocrine mechanisms of reproduction in bivalve molluscs. To gain insights into the molecular control of gonadic development of these animals, G protein-coupled receptors (GPCR) expressed in the gonad of the pacific oyster Crassostrea gigas were investigated. One such receptor was cloned by RT-PCR using oligonucleotide primers derived from consensus sequences of various vertebrate (neuro)peptide receptors. This receptor named Cg-GnRH-related receptor ( Cg GnRH-R) exhibits a high degree of amino acid sequence identity with both vertebrate GnRH receptors and insect AKH receptors. Quantitative RT-PCR shows a specific expression of Cg-GnRH-R in both male and female gonads during the reproductive cycle. This demonstrates for the first time the plausible involvement of a GnRH receptor orthologue in the control of reproduction in a protostomian invertebrate.
Keywords: Mollusc; Crassostrea gigas; Reproduction; Gonad; G Protein-coupled receptor; Gonadotropin-releasing hormone receptor; AKH receptor
Exogenous auxin enhances the degradation of a light down-regulated and nuclear-localized OsiIAA1, an Aux/IAA protein from rice, via proteasome by Jitendra K. Thakur; Mukesh Jain; Akhilesh K. Tyagi; Jitendra P. Khurana (pp. 196-205).
Auxin regulates many aspects of plant growth and development by altering the expression of diverse genes. Among these, the early auxin-responsive genes of Aux/IAA class have been extensively studied in dicots but little information is available on monocots. Earlier, we reported the isolation of OsiIAA1 cDNA, first monocot member of Aux/IAA gene family from rice. Extending this work further, we have isolated the OsiIAA1 gene from rice localized on chromosome 3. The transcriptional start site was mapped to 158 bp upstream to the translational start site. The increased accumulation of OsiIAA1 transcript in auxin-treated rice coleoptiles even in the presence of a protein synthesis inhibitor, cycloheximide, suggested that OsiIAA1 is a primary auxin response gene; the expression of OsiIAA1 gene was also upregulated in the presence of cycloheximide alone. The OsiIAA1 transcript levels were down-regulated in etiolated rice coleoptiles irradiated with far-red, red and blue light, suggesting the existence of a cross-talk between auxin and light signaling. The antibodies raised against His6–OsiIAA1 recombinant protein could detect the OsiIAA1 protein in the plant extract only in the presence of a proteasome inhibitor, MG132, indicating that OsiIAA1 is rapidly degraded by proteasome complex. The degradation of the protein was enhanced by the application of exogenous auxin. Also, the proteasome inhibitor MG132 stabilized the purified His6–OsiIAA1 protein to some extent in the cell-free extracts of rice coleoptiles. The OsiIAA1 protein harbors two nuclear localization signals (NLSs), one bipartite and the other resembling SV40 type NLS. Although both the NLSs were able to target the protein to the nucleus, the bipartite NLS was more effective. These studies indicate that nuclear localization of OsiIAA1 could be a prerequisite for its role in auxin signal transduction.
Keywords: Aux/IAA (gene); Nuclear localization; Proteasome; Auxin signal transduction; Light-hormone interaction
VanabinP, a novel vanadium-binding protein in the blood plasma of an ascidian, Ascidia sydneiensis samea by Masao Yoshihara; Tatsuya Ueki; Takahiro Watanabe; Nobuo Yamaguchi; Kei Kamino; Hitoshi Michibata (pp. 206-214).
Some ascidians accumulate high levels of the transition metal vanadium in their blood cells. The process of vanadium accumulation has not yet been elucidated. In this report, we describe the isolation and cDNA cloning of a novel vanadium-binding protein, designated as VanabinP, from the blood plasma of the vanadium-rich ascidian, Ascidia sydneiensis samea. The predicted amino acid sequence of VanabinP was highly conserved and similar to those of other Vanabins. The N-terminus of the mature form of VanabinP was rich in basic amino acid residues. VanabinP cDNA was originally isolated from blood cells, as were the other four Vanabins. However, Western blot analysis revealed that the VanabinP protein was localized to the blood plasma and was not detectable in blood cells. RT-PCR analysis and in situ hybridization indicated that the VanabinP gene was transcribed in some cell types localized to peripheral connective tissues of the alimentary canal, muscle, blood cells, and a portion of the branchial sac. Recombinant VanabinP bound a maximum of 13 vanadium(IV) ions per molecule with a Kd of 2.8×10−5 M. These results suggest that VanabinP is produced in several types of cell, including blood cells, and is immediately secreted into the blood plasma where it functions as a vanadium(IV) carrier.
Keywords: Ascidian; Vanadium; Metal accumulation; Blood plasma
Tissue-specific and glucose-responsive expression of the pancreatic derived factor (PANDER) promoter by Brant R. Burkhardt; Michael C. Yang; Claudia E. Robert; Scott R. Greene; K. Kelly McFadden; Jichun Yang; Jianmei Wu; Zhiyong Gao; Bryan A. Wolf (pp. 215-225).
Pancreatic derived factor (PANDER) is a recently identified cytokine-like protein that is dominantly expressed in the islets of Langerhans of the pancreas. To investigate the mechanism of tissue-specific regulation of PANDER, we identified and characterized the promoter region. The transcriptional start site was identified 520 bp upstream of the translational start codon by 5′-RLM-RACE. Computer algorithms identified several islet-associated and glucose-responsive binding motifs that included A and E boxes, hepatocyte nuclear factors 1 and 4, Oct-1, and signal transducer and activator of transcription 3, and 5. Reporter gene analysis revealed cell type-specific PANDER promoter expression in islet and liver-derived cell lines. Levels of PANDER mRNA were directly concordant to the observed cell type-specific PANDER promoter gene expression. The minimal element was mapped to the 5′-UTR and located between +200 and +491 relative to the transcriptional start site and imparted maximal gene expression. In addition, several putative glucose-responsive binding sites were further functionally characterized to reveal critical regulatory elements of PANDER. The PANDER promoter was demonstrated to be glucose-responsive in a dose-dependant manner in murine insulinoma β-TC3 cells and primary murine islets, but unresponsive in glucagon-secreting α-TC3 cells. Our findings revealed that the 5′-UTR of PANDER contains the minimal element for gene expression and imparts both tissue-specificity and glucose-responsiveness. The regulation of PANDER gene expression mimics that of insulin and suggests a potential biological function of PANDER involved in metabolic homeostasis.
Keywords: Abbreviations; PANDER; PANcreatic DERived factor; 5′ RLM-RACE; RNA ligase mediated-rapid amplification of cDNA ends; RT-PCR; reverse transcription-polymerase chain reaction; DMEM; Dulbecco's modified Eagle's medium; CMV; cytomegalovirus; EGFP; enhanced green fluorescent protein; ANOVA; analysis of variance; 5′-UTR; 5′-untranslated regionPANDER; Glucose; Promoter; β-TC3; Islet; Transfection
Histone H4 HDAC activity is necessary for expression of the PU.1 gene by R. Nicholas Laribee; Michael J. Klemsz (pp. 226-234).
Although the current paradigm delegates histone deacetylases (HDACs) to the role of transcriptional co-repressors, we recently showed that HDAC activity was necessary for expression of the hematopoietic transcription factor PU.1. Chromatin immunoprecipitation analyses showed that inhibition of HDACs resulted in increased histone H4 acetylation within the promoter and intron 1 regions of the PU.1 locus. In contrast, increases in both H3 and H4 acetylation were seen for introns 2, 3 and 4 on the 3′ end of the PU.1 locus. Maximal increases in histone H4 acetylation over the promoter and intron 1 region were seen within 10 min of HDAC inhibition, while the increases seen on the 3′ end showed slower kinetics. The increases in H4 acetylation were reversible and decreased levels of acetylation correlated with re-expression of the PU.1 gene. Finally, we show that HDAC activity is required for association of RNA polymerase II with the PU.1 promoter.
Keywords: Histone deacetylase; Acetylation; Inhibition
Genomic structures and characterization of the 5′-flanking regions of acyl carrier protein and Δ4-palmitoyl-ACP desaturase genes from Coriandrum sativum by Mi Jung Kim; Jeong Sheop Shin; Jeong-Kook Kim; Mi Chung Suh (pp. 235-244).
The seed-specific or seed-predominant promoters of acyl carrier protein ( Cs-ACP1) and Δ4-palmitoyl-acyl carrier protein desaturase ( Cs-4PAD) genes, which are involved in the biosynthesis of petroselinic acid, were isolated from coriander ( Coriandrum sativum) and analyzed in coriander endosperms and transgenic Arabidopsis. The expression of Cs-ACP1 and Cs-4PAD genes was coordinately regulated during seed development.
Keywords: Acyl carrier protein; Cis-element; Coriandrum sativum; Δ; 4; -palmitoyl-ACP desaturase; Petroselinic acid; Seed-specific promoter
Cloning of GLUT3 cDNA from Atlantic cod ( Gadus morhua) and expression of GLUT1 and GLUT3 in response to hypoxia by Jennifer R. Hall; Robert C. Richards; Tyson J. MacCormack; K. Vanya Ewart; William R. Driedzic (pp. 245-252).
A putative facilitative glucose transporter, GLUT3, cDNA was cloned from Atlantic cod. It is ubiquitously expressed, with substantial levels in kidney. The 519 aa protein has the highest sequence identity (66.3%) to grass carp GLUT3. Atlantic cod were exposed to a hypoxic challenge (45% DO2) for 24 h and the effects on GLUT1 and GLUT3 expression assessed. GLUT1 expression in gill is upregulated; however, in spleen, there is a significant decrease in both GLUT1 and GLUT3 expression. The increase in GLUT1 mRNA is considered to be associated with an increased energy demand on gill, whereas, the decrease in gene expression in spleen potentially reflects a general decrease in rates of transcription.
Keywords: Atlantic cod; Glucose transporter; Gadus morhua; GLUT1; GLUT3; Hypoxia
Expression of SERK family receptor-like protein kinase genes in rice by Yukihiro Ito; Kazuhiko Takaya; Nori Kurata (pp. 253-258).
Some SERK-family receptor-like protein kinase genes have been shown to confer embryonic competence to cells. In this study, we isolated two novel rice genes, OsSERK1 and OsSERK2, belonging to the SERK-family. OsSERK2 showed constitutive expression. The OsSERK1 promoter showed reporter gene activities in some specific tissues in a germinating seed, leaf and root, but not in a developing embryo. This promoter activity suggests that OsSERK1 may have roles in non-embryonic tissues rather than in the embryo.
Keywords: SERK; Receptor-like protein kinase; Embryogenesis; Gene expression; Rice