The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells.

The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.

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An enhancer with cell-type dependent activity is located between the myeloid and epithelial aminopeptidase N (CD 13) promoters

Biochemical Journal ◽

10.1042/bj3220899 ◽

1997 ◽

Vol 322 (3) ◽

pp. 899-908 ◽

Cited By ~ 15

Author(s):

Jørgen OLSEN ◽

Klaus KOKHOLM ◽

Jesper T. TROELSEN ◽

Liselotte LAUSTSEN

Keyword(s):

Transcriptional Start Site ◽

Simian Virus 40 ◽

K562 Cells ◽

Simian Virus ◽

Aminopeptidase N ◽

Cell Type ◽

Independent Manner ◽

Specific Expression ◽

Enhancer Activity ◽

Early Promoter

The 5´ flanking region of the gene encoding the small intestinal brush-border peptidase aminopeptidase N (APN) was screened for the presence of enhancer regions. A 300 bp region with enhancer activity was identified 2.7 kb upstream of the transcriptional start site which is used in epithelial cells. The enhancer stimulated transcription from a heterologous promoter (the simian virus 40 early promoter) in a position- and orientation-independent manner. The activity of the enhancer is cell-type dependent and it is active in liver (HepG2), intestinal (Caco-2) and myeloid (K562) cells. As the epithelial APN promoter is active in the first two cell-types and the myeloid APN promoter in the last, the results may suggest that the enhancer, through a cooperation with either of the promoters, is important for the tissue-specific expression of APN. A detailed analysis of the enhancer led to the identification of four functionally important regions that are protected against DNase I digestion by Caco-2 nuclear extract. Sequence analysis suggests that two of the regions may interact with members of the Ets transcription factor family (Ets is a transformation-specific protein first discovered in the E26 avian erythroblastosis virus), one region with a CCAAT enhancer-binding protein and one region with Sp1, a transcriptional activator first described as a factor binding to the simian virus 40 early promoter.

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Isolation of large T antigen-producing mouse cell lines capable of supporting replication of polyomavirus-plasmid recombinants.

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2406 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2406-2412 ◽

Cited By ~ 27

Author(s):

W J Muller ◽

M A Naujokas ◽

J A Hassell

Keyword(s):

Cell Lines ◽

Tumor Antigens ◽

Simian Virus 40 ◽

Mouse Cell ◽

Simian Virus ◽

Transcription Unit ◽

T Antigen ◽

Coding Region ◽

Large Tumor ◽

Early Promoter

Construction of polyomavirus vectors, analysis of mutant viruses, and rescue of integrated polyomavirus genomes would be considerably aided by the availability of transformed, permissive mouse cell lines capable of producing the viral tumor antigens. To isolate such cell lines, we constructed a hybrid transcription unit composed of the simian virus 40 early promoter fused to the coding region for the polyomavirus tumor antigens. This hybrid transcription unit was used to transform NIH 3T3 cells. Independent foci of transformed cells were isolated, recloned, and characterized. Among 10 lines initially analyzed, 7 supported the replication of origin-bearing plasmid DNAs. Three cell lines were characterized in greater detail. Each line contained one or two independent insertions of polyomavirus DNA and synthesized all three viral tumor antigens. Moreover, the large tumor antigen in two of three lines bound with specificity to sequences about the polyomavirus origin and early promoter. These cell lines should prove useful for studying not only the replication of polyomavirus but also the expression of foreign genes in a mouse cell environment.

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Intestinal Hyperplasia Induced by Simian Virus 40 Large Tumor Antigen Requires E2F2

Journal of Virology ◽

10.1128/jvi.01658-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 13191-13199 ◽

Cited By ~ 20

Author(s):

M. Teresa Sáenz-Robles ◽

Jennifer A. Markovics ◽

Jean-Leon Chong ◽

Rene Opavsky ◽

Robert H. Whitehead ◽

...

Keyword(s):

Transcriptional Repression ◽

Cultured Cells ◽

Simian Virus 40 ◽

Normal Growth ◽

Simian Virus ◽

T Antigen ◽

S Phase ◽

Large Tumor ◽

Intestinal Hyperplasia

ABSTRACT The simian virus 40 large T antigen contributes to neoplastic transformation, in part, by targeting the Rb family of tumor suppressors. There are three known Rb proteins, pRb, p130, and p107, all of which block the cell cycle by preventing the transcription of genes regulated by the E2F family of transcription factors. T antigen interacts directly with Rb proteins and disrupts Rb-E2F complexes both in vitro and in cultured cells. Consequently, T antigen is thought to inhibit transcriptional repression by the Rb family proteins by disrupting their interaction with E2F proteins, thus allowing E2F-dependent transcription and the expression of cellular genes needed for entry into S phase. This model predicts that active E2F-dependent transcription is required for T-antigen-induced transformation. To test this hypothesis, we have examined the status of Rb-E2F complexes in murine enterocytes. Previous studies have shown that T antigen drives enterocytes into S phase, resulting in intestinal hyperplasia, and that the induction of enterocyte proliferation requires T-antigen binding to Rb proteins. In this paper, we show that normal growth-arrested enterocytes contain p130-E2F4 complexes and that T-antigen expression destroys these complexes, most likely by stimulating p130 degradation. Furthermore, unlike their normal counterparts, enterocytes expressing T antigen contain abundant levels of E2F2 and E2F3a. Concomitantly, T-antigen-induced intestinal proliferation is reduced in mice lacking either E2F2 alone or both E2F2 and E2F3a, but not in mice lacking E2F1. These studies support a model in which T antigen eliminates Rb-E2F repressive complexes so that specific activator E2Fs can drive S-phase entry.

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Isolation of large T antigen-producing mouse cell lines capable of supporting replication of polyomavirus-plasmid recombinants

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2406-2412.1984 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2406-2412

Author(s):

W J Muller ◽

M A Naujokas ◽

J A Hassell

Keyword(s):

Cell Lines ◽

Tumor Antigens ◽

Simian Virus 40 ◽

Mouse Cell ◽

Simian Virus ◽

Transcription Unit ◽

T Antigen ◽

Coding Region ◽

Large Tumor ◽

Early Promoter

Construction of polyomavirus vectors, analysis of mutant viruses, and rescue of integrated polyomavirus genomes would be considerably aided by the availability of transformed, permissive mouse cell lines capable of producing the viral tumor antigens. To isolate such cell lines, we constructed a hybrid transcription unit composed of the simian virus 40 early promoter fused to the coding region for the polyomavirus tumor antigens. This hybrid transcription unit was used to transform NIH 3T3 cells. Independent foci of transformed cells were isolated, recloned, and characterized. Among 10 lines initially analyzed, 7 supported the replication of origin-bearing plasmid DNAs. Three cell lines were characterized in greater detail. Each line contained one or two independent insertions of polyomavirus DNA and synthesized all three viral tumor antigens. Moreover, the large tumor antigen in two of three lines bound with specificity to sequences about the polyomavirus origin and early promoter. These cell lines should prove useful for studying not only the replication of polyomavirus but also the expression of foreign genes in a mouse cell environment.

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SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat

Molecular and Cellular Biology ◽

10.1128/mcb.8.1.466-472.1988 ◽

1988 ◽

Vol 8 (1) ◽

pp. 466-472 ◽

Cited By ~ 3

Author(s):

Y Takebe ◽

M Seiki ◽

J Fujisawa ◽

P Hoy ◽

K Yokota ◽

...

Keyword(s):

Leukemia Virus ◽

Expression System ◽

Simian Virus 40 ◽

Simian Virus ◽

Cell Leukemia ◽

Cell Leukemia Virus Type ◽

Early Promoter ◽

Human T Cell ◽

T Cell Leukemia Virus

We developed a novel promoter system, designated SR alpha, which is composed of the simian virus 40 (SV40) early promoter and the R segment and part of the U5 sequence (R-U5') of the long terminal repeat of human T-cell leukemia virus type 1. The R-U5' sequence stimulated chloramphenicol acetyltransferase (CAT) gene expression only when placed immediately downstream of the SV40 early promoter in the sense orientation. The SR alpha expression system was 1 or 2 orders of magnitude more active than the SV40 early promoter in a wide variety of cell types, including fibroblasts and lymphoid cells, and was capable of promoting a high level of expression of various lymphokine cDNAs. These features of the SR alpha promoter were incorporated into the pcD-cDNA expression cloning vector originally developed by Okayama and Berg.

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Activation of an enhancerless gene by chromosomal integration

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4179-4184.1986 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4179-4184

Author(s):

H Hamada

Keyword(s):

Promoter Region ◽

Genomic Dna ◽

Simian Virus 40 ◽

Initiation Site ◽

Simian Virus ◽

Chromosomal Integration ◽

Early Promoter ◽

Sv40 Enhancer ◽

Active Expression ◽

Cat Gene

Expression of enhancerless (E-) and enhancer-containing (E+) genes that are chromosomally integrated was examined. An E- plasmid (pE-cat) containing a chloramphenicol acetyltransferase (cat) gene linked to the simian virus 40 (SV40) early promoter or its E+ counterpart plasmid (pE+-cat) containing the SV40 enhancer was cotransfected into thymidine kinase (TK)-deficient L cells with a cloned tk gene. A number of TK+ transformants were isolated, and expression of the cointegrated cat gene in these cell lines was quantitatively determined by the assay of CAT activity. The results indicated unexpectedly that the E- cat gene was as actively expressed as the E+ cat gene. Analysis of CAT mRNA by primer extension indicated that the E- cat gene, as well as the E+ cat gene, was transcribed from the "native" initiation site contained in the SV40 early promoter region. The active expression of the E- cat gene was maintained in secondary TK+ transformants that arose by transfection with genomic DNA from the primary transformant. These results suggest that expression of the integrated E- cat gene is activated by endogenous enhancer elements.

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Preparation of a "functional library" of African green monkey DNA fragments which substitute for the processing/polyadenylation signal in the herpes simplex virus type 1 thymidine kinase gene

Molecular and Cellular Biology ◽

10.1128/mcb.3.4.643-653.1983 ◽

1983 ◽

Vol 3 (4) ◽

pp. 643-653

Author(s):

G M Santangelo ◽

C N Cole

Keyword(s):

Gene Expression ◽

Thymidine Kinase ◽

Low Frequency ◽

Simian Virus 40 ◽

Simian Virus ◽

African Green Monkey ◽

Dna Fragments ◽

Coding Region ◽

Kinase Gene ◽

Green Monkey

Fragments of African green monkey (Cercopithecus aethiops) DNA (3.5 to 18.0 kilobases) were inserted downstream from the thymidine kinase (TK, tk) coding region in pTK206/SV010, a gene construct which lacks both copies of the hexanucleotide 5'-AATAAA-3' and contains a simian virus 40 origin of replication, allowing it to replicate in Cos-1 cells. No polyadenylated tk mRNA was detected in Cos-1 cells transfected by pTK206/SV010. The ability of simian DNA fragments to restore tk gene expression was examined by measuring the incorporation of [125I]iododeoxycytidine into DNA in Cos-1 cells transfected by pTK206/SV010 insertion derivatives. tk gene expression was restored by the insertion in 56 of the 67 plasmids analyzed, and the level of expression equaled or exceeded that obtained with the wild-type tk gene in 30 of these. In all plasmids examined that showed restoration of tk gene expression, polyadenylated tk mRNA of discrete size was detected. The sizes of these tk mRNAs were consistent with the existence of processing and polyadenylation signals within the inserted DNA fragments. The frequency with which inserted fragments restored tk gene expression suggests that the minimal signal for processing and polyadenylation is a hexanucleotide (AAUAAA or a similar sequence). LTK- cells were biochemically transformed to TK+ with representative insertion constructs. pTK206/SV010 transformed LTK- cells at a very low frequency; the frequency of transformation with insertion derivatives was 40 to 12,000 times higher.

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Hormonally mediated negative regulation of human pro-opiomelanocortin gene expression after transfection into mouse L cells.

Molecular and Cellular Biology ◽

10.1128/mcb.5.9.2443 ◽

1985 ◽

Vol 5 (9) ◽

pp. 2443-2453 ◽

Cited By ~ 30

Author(s):

A Israel ◽

S N Cohen

Keyword(s):

Simian Virus 40 ◽

Simian Virus ◽

Negative Regulation ◽

Coding Region ◽

Transient Expression Assay ◽

S1 Nuclease ◽

Protein Coding ◽

Pomc Gene ◽

Simplex Virus ◽

Nuclease Mapping

We report results indicating that expression and hormonally controlled negative regulation of the human pro-opiomelanocortin (POMC) gene in mouse fibroblasts can be accomplished by the placement nearby of a simian virus 40 enhancer sequence. Expression resulting from correctly initiated transcription required the enhancer in cis both in cells stably transfected with the POMC gene and in a transient expression assay with constructs that fused that POMC promoter region to the protein-coding region of the herpes simplex virus thymidine kinase (TK) gene. Negative regulation of POMC transcription by glucocorticoids was demonstrated in transiently infected cells by assaying for TK activity encoded by the POMC-TK fusion constructs and by quantitative S1 nuclease mapping. The sequences responsible for such regulation were shown to be contained within a DNA segment that extends 670 base pairs upstream from the cap site for POMC mRNA.

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The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer

Molecular and Cellular Biology ◽

10.1128/mcb.8.1.62-70.1988 ◽

1988 ◽

Vol 8 (1) ◽

pp. 62-70

Author(s):

J B Jaynes ◽

J E Johnson ◽

J N Buskin ◽

C L Gartside ◽

S D Hauschka

Keyword(s):

Creatine Kinase ◽

Cultured Cells ◽

Simian Virus 40 ◽

Regulatory Elements ◽

Simian Virus ◽

Major Influence ◽

Transcriptional Enhancer ◽

Muscle Creatine Kinase ◽

Kinase Gene ◽

Muscle Creatine

Muscle creatine kinase (MCK) is induced to high levels during skeletal muscle differentiation. We have examined the upstream regulatory elements of the mouse MCK gene which specify its activation during myogenesis in culture. Fusion genes containing up to 3,300 nucleotides (nt) of MCK 5' flanking DNA in various positions and orientations relative to the bacterial chloramphenicol acetyltransferase (CAT) structural gene were transfected into cultured cells. Transient expression of CAT was compared between proliferating and differentiated MM14 mouse myoblasts and with nonmyogenic mouse L cells. The major effector of high-level expression was found to have the properties of a transcriptional enhancer. This element, located between 1,050 and 1,256 nt upstream of the transcription start site, was also found to have a major influence on the tissue and differentiation specificity of MCK expression; it activated either the MCK promoter or heterologous promoters only in differentiated muscle cells. Comparisons of viral and cellular enhancer sequences with the MCK enhancer revealed some similarities to essential regions of the simian virus 40 enhancer as well as to a region of the immunoglobulin heavy-chain enhancer, which has been implicated in tissue-specific protein binding. Even in the absence of the enhancer, low-level expression from a 776-nt MCK promoter retained differentiation specificity. In addition to positive regulatory elements, our data provide some evidence for negative regulatory elements with activity in myoblasts. These may contribute to the cell type and differentiation specificity of MCK expression.

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