ognizant Communication Corporation

GENE EXPRESSION

ABSTRACTS
VOLUME 9, NUMBER 3, 2000

Gene Expression, Vol. 9, pp. 93-101, 2000
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Copyright © 2000 Cognizant Comm. Corp.
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The 5´Repeat Elements of the Mouse Xist Gene Inhibit the Transcription of X-Linked Genes

Nathalie Allaman-Pillet, Assia Djemaï, Christophe Bonny, and Daniel F. Schorderet

Division of Medical Genetics and Unit of Molecular Genetics, CHUV, Lausanne, Switzerland

X chromosome inactivation in mammals requires the Xist gene, which is exclusively expressed from the inactive X chromosome (Xi). The large heterogeneous Xist nuclear RNA colocalizes with Xi, most likely through nuclear protein interactions. The 5´ region of the Xist RNA contains a series of well-conserved tandem repeats known to bind heteronuclear proteins in vitro and to enhance human XIST transcription. We show in an in vitro system that the conserved repeat element located in the 5´ region of the mouse Xist gene (Xcr) represses three X-linked genes but has no effect on the autosomal genes Aprt, Ins, and the viral SV40 gene. The repression effect is not mediated by the conserved core sequence (Ccs) of Xcr, but requires the presence of the complete Xcr. This Xcr effect on X-linked genes suggests that Xcr transcript recognizes the genes to be silenced and is involved in the spreading of X inactivation.

Key Words: Xist gene; X inactivation; Transcriptional activity; Conserved tandem repeats

Address correspondence to Nathalie Allaman-Pillet, Ph.D., Unit of Molecular Genetics, Division of Medical Genetics, Center Hospitalier, Universitaire Vaudois, CH-1011 Lausanne, Switzerland. Tel: +41 21 314 33 79; Fax: +41 21 314 33 72; E-mail: Nathalie.Pillet@chuv.hospvd.ch




Gene Expression, Vol. 9, pp. 103-114, 2000
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Copyright © 2000 Cognizant Comm. Corp.
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The Role of NF-kB in the Regulation of the Expression of Wilms Tumor Suppressor Gene WT1

Yijing Chen* and Bryan R. G. Williams

Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195

The Wilms tumor suppressor gene, WT1, plays an important role in genitourinary development and the etiology of Wilms tumor. WT1 has a spatially and temporally defined expression in the developing genitourinary system and in specific cells of the hematopoietic system, but the regulatory pathways that control WT1 expression are not well understood. Recently, members of the NF-kB family of transcription factors have been proposed as potent activators of the murine WT1 promoter through binding to a NF-kB site. Because the human WT1 promoter contains a conserved NF-kB site, we investigated whether NF-kB also regulates the expression of the human WT1 gene. We activated NF-kB through cytokine stimulation or inhibited NF-kB through expression of a NF-kB "super repressor" in WT1 expressing Wilms tumor, renal carcinoma, and erythroleukemia cultures and examined the level of endogenous WT1 gene expression. Although a transfected NF-kB reporter construct was responsive to these manipulations, we found that altering NF-kB activity had no effect on endogenous WT1 expression in the cell types used in our study. We conclude that despite the presence of conserved NF-kB elements in the murine and human WT1 promoters, NF-kB is not required to regulate the expression of the WT1 gene in its natural context.

Key Words: Wilms tumor suppressor gene; WT1; NF-kB; Endogenous gene expression; NF-kB super repressor

Address correspondence to Bryan R. G. Williams, Department of Cancer Biology, Lerner Research Institute NB40, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel: (216) 445-9652; Fax: (216) 444-3164; E-mail: williab@ccf.org
 
*Present address: Department of Genetics, University of North Carolina, Chapel Hill, NC 27599.




Gene Expression, Vol. 9, pp. 115-121, 2000
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Human Parainfluenza Virus Type 3 Upregulates ICAM-1 (CD54) Expression in a Cytokine-Independent Manner

Jing Gao, Suresh Choudhary, Amiya K. Banerjee, and Bishnu P. De

Department of Virology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195

Human parainfluenza virus type 3 (HPIV3) causes bronchiolitis, pneumonia, and croup in newborns and infants. Several studies have implicated intercellular adhesion molecule-1 (ICAM-1) in inflammation during infection by viruses. In this study, we investigated the potential for HPIV3 to induce ICAM-1 in HT1080 cells. FACS analysis showed that HPIV3 strongly induced ICAM-1 expression in these cells. The ICAM-1 induction was significantly reduced when the virions were UV inactivated prior to infection, indicating that ICAM-1 induction was mostly viral replication dependent. Culture supernatant of HPIV3-infected cells induced ICAM-1 at an extremely low level, indicating that virus-induced cytokines played only a minor role in the induction process. Consistent with this, potent inducers of ICAM-1 such as IFN-g, TGF-b, and TNF-a were absent in the culture supernatant, but a significant amount of IFN type I was present. By using U2A cells, which are defective in IFN type I signaling, we confirmed that ICAM-1 induction by HPIV3 occurred in a JAK/STAT signaling-independent manner. These data strongly indicate that HPIV3 induces ICAM-1 directly by viral antigens in a cytokine-independent manner; this induction may play a role in the inflammation during HPIV3 infection.

Key Words: Human parainfluenza virus type 3 (HPIV3); Intercellular adhesion molecule-1 (ICAM-1); Inflammation

Address correspondence to Bishnu P. De, Department of Virology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, NC20, Cleveland, OH 44195. Tel: (216) 445-6941; Fax: (216) 444-0512; E-mail:deb@ccf.org




Gene Expression, Vol. 9, pp. 103-114, 2000
1052-2166/00 $20.00 + .00
Copyright © 2000 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
 
The Drosophila TATA Binding Protein Contains a Strong But Masked Activation Domain

Moonkyoung Um* and James L. Manley

Department of Biological Sciences, Columbia University, New York, NY 10027

TATA binding protein (TBP) is a critical transcription factor involved in transcription by all three RNA polymerases (RNAPs). Studies using in vitro systems and yeast have shown that the C-terminal core domain (CTD) of TBP is necessary and sufficient for many TBP functions, but the significance of the N-terminal domain (NTD) of TBP is still obscure. Here, using transient expression assays in Drosophila Schneider cells, we show that the NTD of Drosophila TBP (dTBP) strongly activates transcription when fused to the GAL4 DNA binding domain (DBD). Strikingly, the activity of the NTD is completely repressed in the context of full-length dTBP. In contrast to the much weaker activation obtained by either full-length dTBP or the dTBP CTD fused to the GAL4 DBD, activation by the NTD is dependent on the presence of GAL4 binding sites and is susceptible to the effects of a dominant negative TFIIB mutant, TFIIBDC202, a property observed previously with certain authentic activation domains. Activation by the NTD, but not full-length dTBP or the CTD, seems to be mediated by the action of a strong activation domain, likely a glutamine-rich region. In conclusion, the dTBP NTD can behave as a very strong activator that is masked in the full-length protein, suggesting possible roles for the dTBP NTD in RNAP II-mediated transcription.

Key Words: TATA binding protein; Drosophila; Activation domain; N-terminal domain

Address correspondence to Dr. James L. Manley, Department of Biological Sciences, Sherman Fairchild Center for Life Sciences, Columbia University, 1212 Amsterdam Avenue, New York, NY 10027. Tel: (212) 854-4647; Fax: (212) 865-8246; E-mail: jlm2@columbia.edu

*Present address: Department of Cell Biology, Harvard Medical School, Boston, MA 02115.




Gene Expression, Vol. 9, pp. 133-143, 2000
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Inhibition of Translation of mRNAs Containing g-Monomethylphosphate Cap Structure in Frog Oocytes and in Mammalian Cells

Yahua Chen, Karthika Perumal, and Ram Reddy

Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030

The g-monomethylphosphate cap structure is found in several eukaryotic small RNAs including nuclear U6, U6atac, 7SK, plant nucleolar U3, and rodent cytoplasmic B2 RNAs. In the case of human U6 snRNA, the 5′ end sequence corresponding to nucleotides 1-25 serves as the capping signal and directs the formation of methylphosphate cap structure. In this study, we show that the U6 RNA capping signal, when introduced at the 5´ end of RNAs, can efficiently direct the methylphosphate cap formation in RNAs of up to 2.7 kb long, as well as in different mRNAs. These data show that the methylphosphate capping signal functions in mRNAs having different primary sequences and different lengths. Presence of the methylphosphate cap structure on the 5´ end of a luciferase mRNA with EMCV 5´ noncoding region, which is translated in an IRES-dependent pathway, resulted in a 6- to 100-fold inhibition of translation compared to the same mRNA with a 5´ triphosphate when microinjected into frog oocytes or expressed in mouse cells in tissue culture. Thus, conversion of the pppG structure to a methyl-pppG structure on the 5′ end of an mRNA, which is translated in an IRES-dependent pathway, results in severe inhibition of translation. These data show that the 5´ end motif of mRNAs plays an important role even in the IRES-mediated mRNA translation.

Key Words: Methylphosphate cap structure; RNA translation; Frog oocytes; Mammalian cells

Address correspondence to Ram Reddy, Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel: (713) 798-7906; Fax: (713) 798-3145; E-mail: rreddy@bcm.tmc.edu



 
Gene Expression, Vol. 9, pp. 145-156, 2000
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Copyright © 2000 Cognizant Comm. Corp.
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An Antiprion Effect of the Anticytoskeletal Drug Latrunculin A in Yeast

Peggy A. Bailleul-Winslett,* Gary P. Newnam, Renee D. Wegrzyn, and Yury O. Chernoff

School of Biology and Institute for Bioengineering and Bioscience, Georgia Institute of Technology, M/C 0363, Atlanta, GA 30332-0363

Prions are infectious aggregation-prone isoforms of the normal proteins, supposedly able to seed aggregation of the normal cellular counterparts. In vitro, prion proteins form amyloid fibers, resembling cytoskeletal structures. Yeast prion [PSI], which is a cytoplasmically inherited aggregated isoform of the translation termination factor Sup35p (eRF3), serves as a useful model for studying mechanisms of prion diseases and other amyloidoses. The previously described interaction between Sup35p and cytoskeletal assembly protein Sla1p points to the possible relationships between prions and cytoskeletal networks. Although the Sup35PSI+ aggregates do not colocalize with actin patches, we have shown that yeast cells are efficiently cured of the [PSI] prion by prolonged incubation with latrunculin A, a drug disrupting the actin cytoskeleton. On the other hand, treatments with sodium azide or cycloheximide, agents blocking yeast protein synthesis and cell proliferation but not disrupting the cytoskeleton, do not cause a significant loss of [PSI]. Moreover, simultaneous treatment with sodium azide or cycloheximide blocks [PSI] curing by latrunculin A, indicating that prion loss in the presence of latrunculin A requires a continuation of protein synthesis during cytoskeleton disruption. The sodium azide treatment also decreases the toxic effect of latrunculin A. Latrunculin A influences neither the levels of total cellular Sup35p nor the levels of chaperone proteins, such as Hsp104 and Hsp70, which were previously shown to affect [PSI]. This makes an indirect effect of latrunculin A on [PSI] via induction of Hsps unlikely. Fluorescence microscopy detects changes in the structure and/or localization of the Sup35PSI+ aggregates in latrunculin A-treated cells. We conclude that the stable maintenance of the [PSI] prion aggregates in the protein-synthesizing yeast cells partly depends on an intact actin cytoskeleton, suggesting that anticytoskeletal treatments could be used to counteract some aggregation-related disorders.

Key Words: Actin; Sup35p; Release factor; [PSI]; Protein aggregation; Sodium azide; Cycloheximide

Address correspondence to Yury O. Chernoff, Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332-0363. Tel: (404) 894-1157; Fax: (404) 894-0519, (404) 894-2291; E-mail: yc22@prism.gatech.edu
 
*Present address: Proteome Pathophysiology Program, Huntington Medical Research Institutes and California Institute of Technology, 99 North El Molino Avenue, Pasadena, CA 91101-1830.