ognizant Communication Corporation



Gene Expression, Vol. 8, pp. 1-18, 1999
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Copyright © 1999 Cognizant Comm. Corp.
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A Multiprotein Complex Consisting of the Cellular Coactivator p300, AP-1/ATF, as well as NF-kB Is Responsible for the Activation of the Mouse Major Histocompatibility Class I (H-2Kb) Enhancer A

Dieter Brockmann, Brigitte M. Pützer, Kai S. Lipinski, Ulla Schmücker, and Helmut Esche

Institute of Molecular Biology (Cancer Research), University of Essen Medical School, Hufelandstra ße 55, 45122 Essen, Germany

Major histocompatibility complex (MHC) class I genes encode highly polymorphic antigens that play an essential role in a number of immunological processes. Their expression is activated in response to a variety of signals and is mediated through several promoter elements among which the enhancer A is one of the key control regions. It contains binding sites for several transcription factors, for example: (i) a well-characterized binding site for rel/NF-kB transcription factors in its 3´-end (the H2TF1 or kB1 element), (ii) a second kB site (the kB2 element), which is located immediately adjacent 5´ to the H2TF1 element and which is recognized by p65/relA in the human HLA system, and (iii) an AP-1/ATF recognition sequence in the 5´ end (EnA-TRE). Here we demonstrate that latter element is bound by at least two distinct heterodimers of the AP-1/ATF transcription factor family, namely c-Jun/ATF-2 and c-Jun/Fra2. Moreover, our data reveal that the enhancer A is simultaneously bound by AP-1/ATF and rel/NF-kB transcription factors and that the cellular coactivator p300, which enhances enhancer A-driven reporter gene expression if cotransfected, is recruited to the enhancer A through this multiprotein complex. In contrast to the complete enhancer A, neither the EnA-TRE nor the H2TF1 element on their own are able to confer activation on a heterologous promoter in response to the phorbol ester tumor promoter TPA or the cytokine TNFa . Moreover, deletion of any one of the enhancer A control elements results in a dramatic loss of its inducibility by TNFa , and point mutations in either the EnA-TRE or the H2TF1 element lead to the loss of AP-1/ATF or NF-kB binding, respectively, and to the loss of enhancer A inducibility. Therefore, we conclude that the enhancer A is synergistically activated through a multiprotein complex containing AP-1/ATF, NK-kB transcription factors as well as the cellular coactivator p300.

Key words: AP-1/ATF; MHC class I; NF-kB; p300

Address correspondence to D. Brockmann, Institute of Molecular Biology (Cancer Research), University of Essen Medical School, Hufelandstraße 55, 45122 Essen, Germany. Tel: 49-201-7233687; Fax: 49-201-7235974; E-mail: dieter.brockmann@uni-essen.de

Gene Expression, Vol. 8, pp. 19-32, 1999
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E1A + cHa-ras Transformed Rat Embryo Fibroblast Cells Are Characterized by High and Constitutive DNA Binding Activities of AP-1 Dimers With Significantly Altered Composition

Tatiana V. Pospelova,1 Alexander V. Medvedev,1 Alexander N. Kukushkin,1 Svetlana B. Svetlikova,1 Alex J. Van Der Eb,2 Josephine C. Dorsman,2 and Valery A. Pospelov1

1Institute of Cytology, Russian Academy of Sciences, Tikhoretzky ave., 4, 194064 St-Petersburg, Russia
2Laboratory for Molecular Carcinogenesis, Sylvius Laboratory, Leiden University, Wassenaarseweg 72, 2333 AL, Leiden, The Netherlands

Transcription factors of the AP-1/ATF family, including c-Fos, c-Jun, and ATF-2, play an important role in the regulation of cell proliferation and differentiation, and changes in their levels and/or activities may contribute to oncogenesis. We analyzed the alterations of AP-1/ATF transcription factors upon immortalization and transformation in a panel of cell lines derived from rat embryo fibroblast (REF) cells. The tumorigenic E1A + cHa-ras cells are characterized by high and constitutive DNA binding activities of AP-1, in contrast to nontransformed cells and the E1A cells. The expression of c-fos and c-jun genes was affected differently by the oncogenic transformation. By using antibodies to c-Jun and c-Fos proteins in electrophoretic mobility shift assays (EMSA), we showed that E1A + cHa-ras transformants did not contain c-Fos under any condition of cell cultivation and growth factor stimulation, whereas c-Jun was constitutively upregulated. In the absence of c-fos gene expression, c-Fos protein appears to be replaced by proteins of Fos family (Fra-1) and ATF family (ATF-2 and ATFa). To determine the possible mechanisms of c-fos downregulation in E1A + cHa-ras transformants we have obtained populations of geneticin-resistant clones containing integrated reporter construct -711fos-CAT and its mutants in serum-responsive element (SRE) and cAMP-responsive element (CRE). Data obtained show that the mutations within the SRE lead to a manifold activation of fos-CAT expression. This allows to suggest that c-fos downregulation in E1A + cHa-ras transformants is provided by a negative control mediated through the SRE regulatory region. The profound differences in regulation and composition of transcription factors of the AP-1 family probably play a pivotal role in the transformation of REF cells by E1A and cHa-ras oncogenes.

Key words: E1A and cHa-ras oncogenes; fos and jun expression; AP-1 transcription factors

Address correspondence to Valery A. Pospelov, Institute of Cytology, Russian Academy of Sciences, Tikhoretzky ave., 4, 194064 St-Petersburg, Russia. Tel: (812) 247-1816; Fax: (812) 247-0341; E-mail: vap@link.cytspb.rssi.ru

Gene Expression, Vol. 8, pp. 33-42, 1999
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Autonomously Binding Protein Detected on ets Box of c-fos Serum Response Element in Proliferating Cells

Hiroshi Masutani,* Laura Magnaghi-Jaulin,** Regina Groisman,*** Slimane Ait-Si-Ali,* Philippe Robin,** LindaL. Pritchard,** and Annick Harel-Bellan**

Laboratoire de Biologie des Tumeurs Humaines, CNRS URA 1156, Institut Gustave Roussy, 94805 Villejuif Cedex, France

The serum response element (SRE) in the c-fos promoter contains an ets box whose integrity is required for full activation of this proto-oncogene by nerve growth factor (NGF) in PC12 rat pheochromocytoma cells. Electrophoretic mobility shift assays (EMSA) detect a protein in nuclear extracts that binds to the wild-type SRE, but not to an SRE containing a mutated ets box. Competition studies using unlabeled probes, and supershift experiments using antibodies and in vitro translated core serum response factor (SRF) indicate that the protein in question is not YY1, SAP-1, nor Elk-1 and that it does not exhibit ternary complex factor (TCF) activity, so that it may correspond to an autonomously binding Ets family protein. The complete disappearance of this "Ets-like autonomous binding factor'' upon terminal differentiation of both L6a 2 myoblastic and PC12 pheochromocytoma cells points to a possible role in the proliferation/differentiation process.

Key words: PC12; c-fos; Serum response element (SRE); Ets; Nerve growth factor (NGF)

Address correspondence to Annick Harel-Bellan at her present address: CNRS UPR 9079, IFC 01, 7 rue Guy Moquet, B.P. 8, 94801 Villejuif Cedex, France. Tel: 33 (0)1 4958 3385; Fax: 33 (0)1 4958 3307; E-mail: ahbellan@vjf.cnrs.fr
*Present address: Department of Biological Responses, Institute for Virus Research, Kyoto University, 53 Shogoin, Kawahara-cho, Sakyo-Ku, Kyoto 606, Japan.
**Present address: CNRS UPR 9079, IFC 01, 7 rue Guy Moquet, D.P. 8, 94801 Villejiuf Cedex, France.
***Present address: Departmen tof Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115.

Gene Expression, Vol. 8, pp. 43-57, 1999
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Functional Analysis of the Ume3p/Srb11p-RNA Polymerase II Holoenzyme Interaction

Katrina F. Cooper and Randy Strich

Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111

The yeast C-type cyclin Ume3p/Srb11p and its cyclin-dependent kinase (Cdk) Ume5p are required for the full repression of genes involved in the stress response or meiosis. This cyclin-Cdk kinase copurifies with the RNA polymerase II holoenzyme complex, suggesting it functions through modification of the transcriptional machinery. This report describes two domains required for Ume3p-RNA Pol II holoenzyme association. One domain contains the highly conserved cyclin box that directs cyclin-Cdk interaction and requires Ume5p for holoenzyme binding. The second domain, termed HAD for holoenzyme associating domain, is located within the amino-terminal region of the cyclin and is sufficient for holoenzyme binding independent of Ume5p or the cyclin box. In addition to its role in RNA Pol II holoenzyme association, the HAD is also required for Ume3p-dependent repression in vivo. Finally, HAD mutations do not affect the ability of the Ume3p-Ume5p kinase to phosphorylate in vitro the carboxy-terminal domain (CTD) of RNA polymerase II, a reported target of cyclin C-Cdk activity. In conclusion, this study demonstrates that the association of the Ume3p to the holoenzyme is complex, involving two independent domains, both of which are required for full Ume3p-dependent repression in vivo. Furthermore, HAD-dependent repression does not appear to involve CTD phosphorylation, suggesting a different role for this domain in directing Ume3p-Ume5p activity.

Key words: Cyclin C; RNA polymerase II holoenzyme; Protein-protein interaction; Leucine zipper

Address correspondence to Randy Strich, Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel: (215) 728-5321; Fax: (215) 728-3616; E-mail R_Strich@fccc.edu

Gene Expression, Vol. 8, pp. 59-66, 1999
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SM-20 Is a Novel Growth Factor-Responsive Gene Regulated During Skeletal Muscle Development and Differentiation

Maria C. Moschella,1 Keon Menzies,1 Lana Tsao,1* Mark A. Lieb,1 Jhumku D. Kohtz,4 D. Stave Kohtz,2 and Mark B. Taubman1,3

1The Michael A. and Zena Wiener Cardiovascular Institute, Department of Medicine, 2the Department of Pathology, and 3the Department of Physiology, The Mount Sinai School of Medicine, New York, NY 10029
4Skirball Institute, New York University School of Medicine, New York, NY 10016

SM-20 is a novel, evolutionarily conserved "early response'' gene originally cloned from a rat aortic smooth muscle cell (SMC) cDNA library. SM-20 encodes a cytoplasmic protein, which is induced by platelet-derived growth factor and angiotensin II in cultured SMC and is upregulated in intimal SMC of atherosclerotic plaques and injured arteries. We have now examined SM-20 expression during differentiation of cultured skeletal myoblasts and during skeletal myogenesis in vivo. Low levels of SM-20 mRNA and protein were expressed in proliferating mouse C2C12 myoblasts. Differentiation by serum withdrawal was associated with a marked induction of SM-20 mRNA and the expression of high levels of SM-20 antigen in myotubes. The induction was partially inhibited by blocking differentiation with bFGF or TGFb. Similar results were obtained with the nonfusing mouse C25 myoblast line, suggesting that SM-20 upregulation is a consequence of biochemical differentiation and is fusion independent. During mouse embryogenesis, SM-20 was first observed at 8.5E in the dermomyotomal cells of the rostral somites. SM-20 expression progressed in a rostral to caudal pattern, with highest levels seen in the muscle primordia and mature muscles. SM-20 thus represents a novel intracellular protein that is regulated during skeletal muscle differentiation and development.

Key words: Skeletal muscle; Mouse embryo; Myogenesis; SM-20; Myocyte differentiation; Antibody staining; Somites

Address correspondence to Mark B. Taubman, M.D., Box 1269, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Tel: (212) 241-0047; Fax: (212) 860-7032; E-mail: m_taubman@smtplink.mssm.edu

Gene Expression, Vol. 8, pp. 67-84, 1999
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Regulation of HIV-1 Transcription

Kenneth A. Roebuck and Mohammed Saifuddin

Department of Immunology/Microbiology, Rush Presbyterian St. Luke's Medical Center, Chicago, IL 60612

Human immunodeficiency virus type-1 (HIV-1) is a highly pathogenic lentivirus that requires transcription of its provirus genome for completion of the viral life cycle and the production of progeny virions. Since the first genetic analysis of HIV-1 in 1985, much has been learned about the transcriptional regulation of the HIV-1 genome in infected cells. It has been demonstrated that HIV-1 transcription depends on a varied and complex interaction of host cell transcription factors with the viral long terminal repeat (LTR) promoter. The regulatory elements within the LTR interact with constitutive and inducible transcription factors to direct the assembly of a stable transcription complex that stimulates multiple rounds of transcription by RNA polymerase II (RNAPII). However, the majority of these transcripts terminate prematurely in the absence of the virally encoded trans-activator protein Tat, which stimulates HIV-1 transcription elongation by interacting with a stem-loop RNA element (TAR) formed at the extreme 5' end of all viral transcripts. The Tat-TAR interaction recruits a cellular kinase into the initiation-elongation complex that alters the elongation properties of RNAPII during its transit through TAR. This review summarizes our current knowledge and understanding of the regulation of HIV-1 transcription in infected cells and highlights the important contributions human lentivirus gene regulation has made to our general understanding of the transcription process.

Key words: HIV-1; Transcription factors; Long terminal repeat; Tat; Chromatin; Promoter; Nuclear factor kappa B (NF-kB); Activator protein-1

Address correspondence to Kenneth A. Roebuck, Department of Immunology/Microbiology, Rush Presbyterian St. Luke's Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612. Fax: (312) 942-2808; E-mail:kroebuck@rush.edu

Gene Expression, Vol. 8, pp. 85-103, 1999
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Dual Tandem Promoter Elements Containing CCAC-Like Motifs From the Tetrodotoxin-Resistant Voltage-Sensitive Na+ Channel (rSkM2) Gene Can Independently Drive Muscle-Specific Transcription in L6 Cells

Hui Zhang,1 Michelle N. Maldonado,1 Robert L. Barchi,2,3 and Roland G. Kallen1,3

Department of 1Biochemistry and Biophysics, 2Neurology and Neuroscience, and the 3David Mahoney Institute of Neurological Sciences, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

cis-Elements in the -129/+124 promoter segment of the rat tetrodotoxin-resistant voltage-gated sodium channel (rSkM2) gene that are responsible for reporter gene expression in cultured muscle cells were identified by deletion and scanning mutations. Nested 5' deletion constructs, assayed in L6 myotubes and NIH3T3 cells, revealed that the minimum promoter allowing muscle-specific expression is contained within the -57 to +1 segment relative to the major transcription initiation site. In the context of the -129/+1 construct, however, scanning mutations in the -69/+1 segment failed to identify any critical promoter elements. In contrast, identical mutations in a minimal promoter (-57/+124) showed that all regions except -29/-20 are essential for expression, especially the -57/-40 segment, consistent with the 5' deletion analysis. Further experiments showed that the distal (-129/-58) and proximal promoter (-57/+1) elements can independently drive reporter expression in L6 myotubes, but not in NIH3T3 fibroblasts. This pair of elements is similar in sequence and contains Sp1 sites (CCGCCC), CCAC-like motifs, but no E-boxes or MEF-2 sites. The two segments form similarly migrating complexes with L6 myotube nuclear extracts in gel-shift assays. Critical elements within the distal promoter element were defined by 10 base pair scanning mutations in the -119 to -60 region in the context of the -129/+1 segment containing a mutated -59/-50 segment that inactivates the proximal promoter. Nucleotides in the -119/-90 region, especially -109/-100, were the most important regions for distal promoter function. We conclude that the -129/+1 segment contains two tandem promoter elements, each of which can independently drive muscle-specific transcription. Supershifts with antibodies to Sp1 and myocyte nuclear factor (MNF) implicate the involvement of Sp1, MNF, and other novel factors in the transcriptional regulation of rSkM2 gene expression.

Key words: Sodium channel; Skeletal muscle; Expression; cis-Element; L6 myotubes; Electrophoresis mobility shift analysis

Address correspondence to Roland G. Kallen, M.D., Ph.D., Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 913B Stellar-Chance Bldg., 422 Curie Blvd., Philadelphia, PA 19104-6059. Tel: (215) 898-5184; Fax: (215) 573-7058; E-mail: rgk@mbio.med.upenn.edu

Gene Expression, Vol. 8, pp. 105-114, 1999
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Two Internal Sequence Elements Modulate Transcription From the External Human 7S K RNA Gene Promoter In Vivo

Bjorn Sandrock* and Bernd-Joachim Benecke

Department of Biochemistry NC6, Faculty of Chemistry, Ruhr University, D-44780 Bochum, Germany

Constructs of the external promoter of the human 7S K RNA gene in combination with different reporter elements and varying amounts of 7S K internal sequences were analyzed for efficient transcription by RNA polymerase III (pol III) in vitro and in vivo. In vitro, the 7S K promoter alone (-245 to -1) revealed full activity, compared to the entire wild-type gene. In vivo, however, the activity of the gene-external 7S K promoter, albeit clearly functional by itself, was positively modulated by internal sequence elements. Fusion constructs containing increasing amounts of transcribed 7S K sequences revealed that two elements were responsible for this activation. One element is associated with the initiator region (+1 to +8) of this class III gene. The second sequence comprises the 5' half of a cryptic A-box starting at +10 of the 7S K RNA sequence. In the context of a totally unrelated vector sequence, a GGC element alone was sufficient to functionally replace that cryptic A-box. Thus, it appears that in context of the 7S K RNA gene--and possibly the 7S L and U6 RNA genes as well--structurally divergent A-box-like elements function as internal modulators of these pol III promoters.

Key words: 7S K promoter; Internal elements; Polymerase III; External promoter

*Present address: IGBMC, B.P. 163, F-67404 Illkirch Cedex, France.
Address correspondence to Bernd-Joachim Benecke. Tel: (++49) 234 700-4233; Fax (++49) 234 709-4244.

Gene Expression, Vol. 8, pp. 115-127, 1999
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Constitutive and Inducible In Vivo Protein-DNA Interactions at the Tumor Necrosis Factor-a Promoter in Primary Human T Lymphocytes

Christoph Becker,1 Karina Barbulescu,1 Stefan Wirtz,1 Karl-Hermann Meyer zum Buschenfelde,1 Sven Pettersson,2 and Markus F. Neurath1

1Laboratory of Immunology, I. Medical Clinic, University of Mainz, Germany
2Center of Genomics Research, Karolinska Institute, Stockholm, Sweden

Tumor necrosis factor-a (TNF-a) is a key cytokine of lymphocytes with major regulatory functions in immunomodulation, chronic inflammation, and septic shock. However, only limited information on TNF promoter regulation in vivo in primary lymphocytes is available. To determine and compare protein-DNA interactions at the native TNF locus in primary lymphocytes, we analyzed the human TNF-a promoter by ligation-mediated polymerase chain reaction (LM-PCR) techniques. Accordingly, primary CD4+ T lymphocytes from peripheral blood were cultured in the presence of various stimuli and analyzed by LM-PCR. Inducible in vivo protein-DNA interactions at the TNF promoter were detected between -120 and -70 bp of the human TNF promoter relative to the transcriptional start site. This area includes binding sites for transcription factors such as ETS-1, NFAT, ATF-2/c-jun, SP-1/Egr-1, and NF-kB. In contrast, no protein-DNA interactions were observed at various binding sites with reported regulatory function in tumor cell lines such as the k2 element, the NFAT site at -160, the AP1 site at -50, and the SP1 site at -65. Additional mutagenesis and transfection studies demonstrated that NF-kB and CREB/ AP-1 are important regulators of inducible TNF promoter activity in primary human T lymphocytes. These results provide novel insights into the complex regulation of TNF gene transcription in primary T lymphocytes in vivo by constitutive and inducible protein-DNA interactions that appear to be at least partially different compared to previously characterized tumor cell lines.

Key words: TNF; Promoter; In vivo footprinting; AFT-2/c-jun; SP1/Egr

Address correspondence to Markus F. Neurath, M.D., Laboratory of Immunology, I. Medical Clinic, University of Mainz, Langenbeckstr. 1, 55131 Mainz, Germany. Tel: 0049-6131-173382; Fax: 0049-6131-175508.

Gene Expression, Vol. 8, pp. 129-139, 1999
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Changes in Levels of Normal ML-1 Gene Transcripts Associated With the Conversion of Human Nontumorigenic to Tumorigenic Phenotypes

Xiao Li Sun,1 Dawei Li,2 Jin Fang,1 Inge Noyes,1 Bruce Casto,1 Karl Theil,4 Charles Shuler,3 and George E. Milo1

1Department of Medical Biochemistry and Comprehensive Cancer center, The Ohio State University College of Medicine and Public Health, Columbus, OH
2Harvard University, College of Medicine, Boston, MA
3Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA
4Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH

Evaluation of malignant human tumors in a xenobiotic nude mouse system has demonstrated that not all cells in tumors exhibit the capacity to form progressively growing tumors. However, nontumorigenic cells isolated from human tumors can be converted to a tumorigenic phenotype in nude mice by treatment with chemical carcinogens or by transfection with antisense to tumor suppressor genes. A newly discovered gene, designated ML-1, appears to be associated with tumorigenesis, because an ML-1 antisense cDNA construct, transfected into nontumorigenic, anchorage-independent growth (AIG) cells, was sufficient to convert these cells into a tumorigenic phenotype. The AIG cells transfected with ML-1 antisense cDNA constructs and converted to tumorigenic cells did not exhibit expression of normal ML-1 mRNA transcripts in the converted cells when evaluated by Northern analysis, whereas premalignant and normal cells expressed ML-1 transcripts at a high level. The converted cells exhibited a loss of growth control and produced tumors in a surrogate nude mouse that were greater than 2.0 cm in less than 2 months. The ML-1 gene has a DNA sequence that is 2177 bp in size and is located on chromosome number 13 on the q arm at site 12-14. Sequence analysis and investigation of GenBank sequences indicate that this is a newly described human gene.

Key words: ML-1; Malignant conversion; Antisense cDNA; Chromosome 13

Address correspondence to George E. Milo, College of Medicine, Department of Medical Biochemistry, 1645 Neil Avenue, Room 410 Hamilton Hall, The Ohio State University, Columbus, OH 43210. Tel: (614) 292-1478; Fax: (614) 292-7032; E-mail: milo.1@osu.edu

Gene Expression, Vol. 8, pp. 141-149, 1999
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The T7 Concatemer Junction Sequence Interferes With Expression From a Downstream T7 Promoter In Vivo

Bohdan Harvey,1 Malgorzata Korus,2 and Emanuel Goldman1,2

1Graduate School of Biomedical Sciences, 2New Jersey Medical School, Department of Microbiology & Molecular Genetics, University of Medicine & Dentistry of New Jersey, Newark, NJ

A recently described new signal for transcription termination in vitro by T7 RNA polymerase has now been tested in vivo. This signal, identified during transcription of the cloned human preproparathyroid hormone (PTH) gene, is also found in the phage T7 genome, at the concatemer junction (CJ). We introduced the 17-bp concatemer junction sequence at the ends of a test gene and control gene (both derived from T7 gene 9) in a T7 vector previously used to study effects of rare codons on expression. The CJ elements replaced the original vector's RNase III processing sites, and a new T7 promoter was also introduced to drive the downstream (control) gene. We assayed for test and control gene mRNA and protein by direct labeling with [32P]phosphate and [35S]methionine. The altered vector with CJ sequences (pCT1.1) expressed the upstream test gene, but showed poor expression of the downstream control gene. No discrete T7 mRNA bands could be discerned by direct labeling with 32P. A precursor vector with only the control gene in single copy expressed the protein much better, suggesting that the inhibition of control gene expression in pCT1.1 was a result of the upstream CJ element at the 3' end of the test gene. RT-PCR experiments were consistent with readthrough and possibly pausing at CJ. An RNA folding program predicts a highly stable secondary structure between the upstream CJ element and the control gene's translation start signals. These data support an interpretation that the CJ element is ineffective as a T7 transcription terminator in vivo in this vector, and that structure of the readthrough transcript blocks ribosome access to the downstream translation start. The readthrough transcripts are also likely to be less stable than properly terminated or processed T7 mRNA, because levels of test protein expression in pCT1.1 were reduced compared to original vector, and basal expression was negligible, while the original codon test vector shows substantial basal expression.

Key words: Concatemer junction; T7 promoter; Preproparathyroid hormone

Address correspondence to Emanuel Goldman, Department of Microbiology & Molecular Genetics, University of Medicine & Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07103. Tel: (973) 972-4367; Fax: (973) 972-3644; E-mail: egoldman@umdnj.edu

Gene Expression, Vol. 8, pp. 151-163, 1999
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Far Upstream Regulatory Elements Enhance Position-Independent and Uterus-Specific Expression of the Murine a1(I) Collagen Promoter in Transgenic Mice

Kimberly Krempen,1 Doris Grotkopp,1 Keith Hall,1 Alexandra Bache,1 Andrea Gillan,2 Richard A. Rippe,2 David A. Brenner,2 and Michael Breindl1

1Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, CA
2Department of Medicine and Biochemistry and Biophysics, The University of North Carolina, Chapel Hill, NC

The stage- and tissue-specific expression of many eukaryotic genes is regulated by cis-regulatory elements, some of which are located in proximity to the start site of transcription whereas others have been identified at considerable distances. In previous studies we have identified far upstream DNase I-hypersensitive sites in the murine a1(I) collagen (Col1a1) gene, which may play a role in the regulation of this abundantly expressed gene. Here we have cloned several of these sites into reporter gene constructs containing the Col1a1 promoter driving the green fluorescent protein (GFP) reporter gene and tested their possible functions in transfection experiments and transgenic mice. In transient and stable transfections none of the hypersensitive sites had a significant effect on Col1a1 promoter activity, indicating that they do not contain a classical transcriptional enhancer. In transgenic animals one element located at -\18 to -19.5 kb enhanced the position-independent activity of the linked Col1a1 promoter and may be part of a locus control region. Another element located at -7 to -8 kb specifically enhanced reporter gene expression in the uteri of transgenic mice, suggesting that it contains a novel transcriptional enhancer that may be involved in the regulation of type I collagen expression in tissue remodeling in the uterus during the estrous cycle. Our studies also demonstrate the versatility of the GFP reporter gene for use in transgenic animals because it can be analyzed in live animals, whole mount embryos, histological thin sections, or primary cell cultures, and it can be quantified very sensitively in tissue or cell extracts using a fluorometer.

Key words: a1(I) collagen; Green fluorescent protein; Transgenic mice; Distal regulatory elements; Enhancer; Uterus

Address correspondence to Michael Breindl, Ph.D., Department of Biology, San Diego State University, San Diego, CA 92182. Tel: (619) 594-2983; Fax: (619) 594-5676; E-mail: mbreindl@sunstroke.sdsu.edu

Gene Expression, Vol. 8, pp. 165-174, 1999
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Faithful In Vitro Transcription by Fission Yeast RNA Polymerase III Reveals Unique a-Amanitin Sentivity

Florian Rödicker, Friedrich Ossenbühl, Diemo Michels,* and Bernd-Joachim Benecke

Department of Biochemistry NC6, Ruhr-University, D-44780 Bochum, Germany

Transcription with fission yeast (Schizosaccharomyces pombe) RNA polymerase III (pol III) was studied in two different in vitro systems. Reactions performed with isolated nuclei gave rise to 5S and pre-tRNA molecules. Because the a-amanitin sensitivity of that reaction clearly differed from what has been observed with pol III enzymes of other eukaryotes, a cell-free S. pombe transcription extract was developed and analyzed with the homologous 7S L RNA (srp RNA; signal recognition particle RNA) gene. Synthesis of 7S L RNA was found to be sensitive to high concentrations of a-amanitin, with 50% reduction seen at 400 mg/ml of the toxin. However, even with very high a-amanitin concentrations, exceeding 1 mg/ml, no full inhibition of the S. pombe pol III enzyme could be obtained. Together, these results demonstrate that in contrast to the yeast Saccharomyces cerevisiae, pol III from S. pombe is sensitive to high concentrations of a-amanitin, yet with a clearly different dose response than that observed with the corresponding RNA polymerase of higher eukaryotes. Furthermore, while the S. pombe 7S L RNA gene was efficiently transcribed in HeLa cell extracts, the human 7S L RNA gene was not actively transcribed in the S. pombesystem. This finding of divergent promoter structures of both genes was verified by the analysis of 5' deletion mutants of the S. pombe 7S L RNA gene.

Key words: Schizosaccharomyces pombe; Cell-free extract; RNA polymerase III transcription; \GK\a-Amanitin sensitivity

*Present address: Institut f\u\ur Zellbiologie, University of Essen, Virchowstr.173, D-45122 Essen, Germany.

Address correspondence to Bernd-Joachim Benecke. Tel. ++49 234 700-4233; Fax: ++49 234 709-4244; E-mail: bernd.benecke@rz.ruhr-uni-bochum.de

Gene Expression, Vol. 8, pp. 175-186, 1999
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Regulation of Hepatitis B Virus Expression in Progenitor and Differentiated Cell Types: Evidence for Negative Transcriptional Control in Nonpermissive Cells

Michael Ott, Qiangzhong Ma, Baiquan Li, S. Gagandeep, Leslie E. Rogler, and Sanjeev Gupta

Marion Bessin Liver Research Center, Cancer Research Center and Department of Medicine, Jack and Pearl Resnick Campus, Albert Einstein College of Medicine, Bronx, NY

Mechanisms regulating cell type-specific gene expression are not completely understood. We utilized hepatitis B virus (HBV) enhancer I and preS1 promoter sequences, which exhibit cell type specificity, to analyze transcriptional control in pluripotential murine embryonic stem (ES) cells, bipotential HBC-3 progenitor liver cells, mature hepatocytes, and fibroblasts. In transient transfection assays, HBV sequences were most active in primary hepatocytes, followed by HBC-3 and ES cells, and became inactive in fibroblasts. Cotransfections with HNF-3 or C/EBP plasmids increased expression of HBV sequences in hepatocytes and HBC-3 cells. However, increased HBV expression was not observed in ES cells and HBV remained inactive in fibroblasts, suggesting different transcriptional controls, which was compatible with alterations in the abundance of endogenous transcription factors. Analysis in somatic hybrid cells created from NIH 3T3 fibroblasts and Hepa1-6 mouse hepatocytes with expression of albumin and selected hepatic transcription factors showed that HBV sequences were expressed weakly but without increased expression following transfection of HNF-1, HNF-3, and C/EBP plasmids. These findings indicated that repression of HBV in nonpermissive cells involved inactivation of transcription factor activity. Expression of HBV in stem cells is relevant for mechanisms concerning viral persistence and oncogenesis, as well as analysis of hepatocytic differentiation in progenitor cells.

Key words: Hepatitis B virus; Transcription factor; Gene expression; Stem cell; Liver

Address correspondence to Sanjeev Gupta, M.D., Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Ullmann 625, 1300 Morris Park Avenue, Bronx, NY 10461. Tel: (718) 430-2098; FAX: (718) 430-8975; E-mail: sanjvgupta@pol.net

Gene Expression, Vol. 8, pp. 187-196, 1999
1052-2166/99 $20.00 + .00
Copyright © 1999 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Overexpression, Purification, and Partial Characterization of ADP-Ribosyltransferases ModA and ModB of Bacteriophage T4

Bernd Tiemann, Reinhard Depping, and Wolfgang Rüger

Arbeitsgruppe Molekulare Genetik, Fakultät für Biologie, Ruhr-Universität Bochum,, D-44780 Bochum, Germany

There is increasing experimental evidence that ADP-ribosylation of host proteins is an important means to regulate gene expression of bacteriophage T4. Surprisingly, this phage codes for three different ADP-ribosyltransferases, gene products Alt, ModA, and ModB, modifying partially overlapping sets of host proteins. While gene product Alt already has been isolated as a recombinant protein and its action on host RNA polymerases and transcription regulation have been studied, the nucleotide sequences of the two mod genes was published only recently. Their mode of action in the course of the infection cycle and the consequences of the ADP-ribosylations catalyzed by these enzymes remain to be investigated. Here we describe the cloning of the genes, the overexpression, purification, and partial characterization of ADP-ribosyltransferases ModA and ModB. Both proteins seem to act independently, and the ADP-ribosyl moieties are transferred to different sets of host proteins. While gene product ModA, similarly to the Alt protein, acts also on the a-subunit of host RNA polymerase, the ModB activity serves another set of proteins, one of which was identified as the S1 protein associated with the 30S subunit of the E-coli ribosomes.

Key words: ADP-ribosyltransferase; Lysogeny; ModA; ModB; Alt; Overexpression; Phage T4; Protein purification; Transcription regulation; Translation regulation

Address correspondence to Wolfgang Rüger. Tel: +49 (0) 234 700-3102; Fax: +49 (0) 234 709-4195; E-mail: wolfgang.rueger@ruhr-uni-bochum.de