Target sequences for cis-acting regulation within the dual promoter of the human c-myc gene

1987 ◽  
Vol 7 (4) ◽  
pp. 1393-1400
Author(s):  
M Lipp ◽  
R Schilling ◽  
S Wiest ◽  
G Laux ◽  
G W Bornkamm
Keyword(s):  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Leader Region ◽  
Cis Acting ◽  
Cellular Factors ◽  
Negative Effect ◽  
Myc Gene ◽  
Cellular Transcription ◽  
Cat Gene

Recombinant plasmids of the human c-myc promoter-leader region and the bacterial chloramphenicol acetyltransferase (cat) gene were constructed. After transfection into different rodent and human cells, the 862-base-pair (bp) PvuII fragment carrying both c-myc promoters and 350 bp of the untranslated leader conferred 1/15 to 1/30 of the CAT activity mediated by the simian virus 40 promoter. The presence of additional sequences upstream of the PvuII fragment had an overall negative effect on c-myc promoter activity detectable by titration analysis with small amounts of transfected plasmid DNA. The analysis of numerous deletion constructs in the c-myc promoter-leader region as well as S1 mapping experiments demonstrated that the high CAT activity depended largely on the presence of the second promoter. By cotransfection of c-myc-cat constructs with plasmids carrying different parts of the c-myc promoter locus, targets for positively acting cellular factors were identified. Two positive regulatory elements were mapped within the 862-bp PvuII fragment. One was localized within the 248-bp PvuII-SmaI fragment -101 to -349 bp upstream of the first cap site and the other within the 142-pb XhoI-NaeI fragment of the first exon, comprising positions -95 to +47 relative to the second cap site. We conclude that the dual promotor of the human c-myc gene represents a strong eucaryotic promotor regulated by cooperation of positively and negatively acting cellular transcription factors.

scholarly journals Target sequences for cis-acting regulation within the dual promoter of the human c-myc gene.

1987 ◽  
Vol 7 (4) ◽  
pp. 1393-1400 ◽  
Author(s):  
M Lipp ◽  
R Schilling ◽  
S Wiest ◽  
G Laux ◽  
G W Bornkamm
Keyword(s):  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Leader Region ◽  
Cis Acting ◽  
Cellular Factors ◽  
Negative Effect ◽  
Myc Gene ◽  
Cellular Transcription ◽  
Cat Gene

Recombinant plasmids of the human c-myc promoter-leader region and the bacterial chloramphenicol acetyltransferase (cat) gene were constructed. After transfection into different rodent and human cells, the 862-base-pair (bp) PvuII fragment carrying both c-myc promoters and 350 bp of the untranslated leader conferred 1/15 to 1/30 of the CAT activity mediated by the simian virus 40 promoter. The presence of additional sequences upstream of the PvuII fragment had an overall negative effect on c-myc promoter activity detectable by titration analysis with small amounts of transfected plasmid DNA. The analysis of numerous deletion constructs in the c-myc promoter-leader region as well as S1 mapping experiments demonstrated that the high CAT activity depended largely on the presence of the second promoter. By cotransfection of c-myc-cat constructs with plasmids carrying different parts of the c-myc promoter locus, targets for positively acting cellular factors were identified. Two positive regulatory elements were mapped within the 862-bp PvuII fragment. One was localized within the 248-bp PvuII-SmaI fragment -101 to -349 bp upstream of the first cap site and the other within the 142-pb XhoI-NaeI fragment of the first exon, comprising positions -95 to +47 relative to the second cap site. We conclude that the dual promotor of the human c-myc gene represents a strong eucaryotic promotor regulated by cooperation of positively and negatively acting cellular transcription factors.

Activation of an enhancerless gene by chromosomal integration

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.

Regulation of mouse CYP1A1 gene expression by dioxin: requirement of two cis-acting elements during induction

1989 ◽  
Vol 9 (6) ◽  
pp. 2378-2386
Author(s):  
L A Neuhold ◽  
Y Shirayoshi ◽  
K Ozato ◽  
J E Jones ◽  
D W Nebert
Keyword(s):  
Gene Expression ◽  
Simian Virus 40 ◽  
Class Ii ◽  
Simian Virus ◽  
Class I ◽  
Ah Receptor ◽  
Class Iii ◽  
Cis Acting ◽  
Cyp1a1 Gene ◽  
Sv40 Dna

The mouse cytochrome P1450 (CYP1A1) gene is responsible for the metabolism of numerous carcinogens and toxic chemicals. Induction by the environmental contaminant tetrachlorodibenzo-p-dioxin (TCDD) requires a functional aromatic hydrocarbon (Ah) receptor. We examined the 5'-flanking region of the CYP1A1 gene in mouse hepatoma Hepa-1 wild-type cells and a mutant line having a defect in chromatin binding of the TCDD-receptor complex. We identified two cis-acting elements (distal, -1071 to -901 region; proximal, -245 to -50 region) required for constitutive and TCDD-inducible CYP1A1 gene expression. Three classes of DNA-protein complexes binding to the distal element were identified: class I, found only in the presence of TCDD and a functional Ah receptor, that was heat labile and not competed against by simian virus 40 (SV40) early promoter DNA; class II, consisting of at least three constitutive complexes that were heat stable and bound to SV40 DNA; and class III, composed of at least three constitutive complexes that were thermolabile and were not competed against by SV40 DNA. Essential contacts for these proteins were centered at -993 to -990 for the class I complex, -987, -986, or both for the class II complexes, and -938 to -927 for the class III complexes. The proximal element was absolutely essential for both constitutive and TCDD-inducible CYP1A1 gene expression, and at least two constitutive complexes bound to this region. These data are consistent with the proximal element that binds proteins being necessary but not sufficient for inducible gene expression; interaction of these proteins with those at the distal element was found to be required for full CYP1A1 induction by TCDD.

Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

1988 ◽  
Vol 8 (7) ◽  
pp. 2896-2909 ◽  
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
W M Perry ◽  
D Vizard ◽  
T Weil ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Simian Virus ◽  
Cell Type ◽  
Specific Expression ◽  
Developing Muscle

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

Uniform cell-autonomous tumorigenesis of the choroid plexus by papovavirus large T antigens

1991 ◽  
Vol 11 (12) ◽  
pp. 5968-5976
Author(s):  
J D Chen ◽  
T Van Dyke
Keyword(s):  
Transgenic Mice ◽  
Choroid Plexus ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Simian Virus ◽  
T Antigen ◽  
Rapid Expansion ◽  
Morphological Transformation ◽  
Large Tumor ◽  
Lymphotropic Papovavirus

The simian virus 40 (SV40) large tumor antigen (T antigen) under its natural regulatory elements induces choroid plexus papillomas in transgenic mice. Because these tumors develop focally after several months, it has been suggested that secondary cellular alterations are required to induce a tumor in this tissue. In contrast to SV40, the related lymphotropic papovavirus early region induces rapid nonfocal choroid plexus neoplasia in transgenic mice. Here, using hybrid gene constructs, we showed that T antigen from either virus in in fact sufficient to induce these tumors. Their abilities to induce proliferative abnormalities in other tissues, such as kidney and thymus, were also indistinguishable. Differences in the rate of choroid plexus tumorigenesis reflected differences in the control regions of the two viruses, rather than differences in T antigen per se. Under SV40 regulation, expression was limited to a fraction of the choroid plexus cells prior to the formation of focal tumors. When SV40 T antigen was placed under lymphotropic papovavirus control, in contrast, expression was generally uniform in the choroid plexus and rapid expansion of the tissue ensued. We found a direct relationship between T-antigen expression, morphological transformation, and proliferation of the choroid plexus epithelial cells. Analysis of mosaic transgenic mice indicated further that T antigen exerts its mitogenic effect cell autonomously. These studies form the foundation for elucidating the role of various T-antigen subactivities in tumorigenesis.

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

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.

Late messenger RNA production by viable simian virus 40 mutants with deletions in the leader region

1981 ◽  
Vol 153 (3) ◽  
pp. 589-618 ◽  
Author(s):  
M. Piatak ◽  
K.N. Subramanian ◽  
P. Roy ◽  
S.M. Weissman
Keyword(s):  
Messenger Rna ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Leader Region

scholarly journals Neoplastic transformation of rat 3Y1 cells by a transcriptionally activated human c-myc gene and stabilization of p53 cellular tumor antigen in the transformed cells.

1986 ◽  
Vol 6 (12) ◽  
pp. 4379-4386 ◽  
Author(s):  
K Shiroki ◽  
K Segawa ◽  
Y Koita ◽  
M Shibuya
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Tumor Antigen ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Transformed Cells ◽  
Agar Culture ◽  
Myc Gene ◽  
Tumorigenic Cell Line

Transformed foci were obtained in rat 3Y1 fibroblasts cotransfected with pRmyc 27 (transcriptionally activated c-myc) and pSV2neo DNA. RmycY cell lines (1 to 7) were established from these foci. RmycY cells were small and round and contained enlarged nucleoli in the nucleus. The myc gene was expressed in these cell lines at a much higher level than in 3Y1 cells and at a level similar to that in HL-60 cells. These cell lines formed colonies in soft-agar culture and tumors in syngeneic rats transplanted with RmycY cells. Expression of the gene and colony formation in soft-agar culture were analyzed in subclones from RmycY cell line 1. A correlation between myc gene expression and the ability to form colonies in soft-agar culture was observed in these cells. Antibody against p53 cellular tumor antigen was detected in some sera from tumor-bearing rats. p53 cellular tumor antigen stabilized and accumulated in RmycY cells to the same extent as in simian virus 40-transformed cells. The results suggest that elevated c-myc expression and an increased amount of p53 cause 3Y1 cells to become a more tumorigenic cell line.

scholarly journals Multiple positive and negative 5' regulatory elements control the cell-type-specific expression of the embryonic skeletal myosin heavy-chain gene.

1987 ◽  
Vol 7 (12) ◽  
pp. 4377-4389 ◽  
Author(s):  
P F Bouvagnet ◽  
E E Strehler ◽  
G E White ◽  
M A Strehler-Page ◽  
B Nadal-Ginard ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Chain Gene ◽  
Specific Element

To identify the DNA sequences that regulate the expression of the sarcomeric myosin heavy-chain (MHC) genes in muscle cells, a series of deletion constructs of the rat embryonic MHC gene was assayed for transient expression after introduction into myogenic and nonmyogenic cells. The sequences in 1.4 kilobases of 5'-flanking DNA were found to be sufficient to direct expression of the MHC gene constructs in a tissue-specific manner (i.e., in differentiated muscle cells but not in undifferentiated muscle and nonmuscle cells). Three main distinct regulatory domains have been identified: (i) the upstream sequences from positions -1413 to -174, which determine the level of expression of the MHC gene and are constituted of three positive regulatory elements and two negative ones; (ii) a muscle-specific regulatory element from positions -173 to -142, which restricts the expression of the MHC gene to muscle cells; and (iii) the promoter region, downstream from position -102, which directs transcription initiation. Introduction of the simian virus 40 enhancer into constructs where subportions of or all of the upstream sequences are deleted (up to position -173) strongly increases the level of expression of such truncated constructs but without changing their muscle specificity. These upstream sequences, which can be substituted for by the simian virus 40 enhancer, function in an orientation-, position-, and promoter-dependent fashion. The muscle-specific element is also promoter specific but does not support efficient expression of the MHC gene. The MHC promoter in itself is not muscle specific. These results underline the importance of the concerted action of multiple regulatory elements that are likely to represent targets for DNA-binding-regulatory proteins.

scholarly journals Transcriptional control of the mouse alpha 2(I) collagen gene: functional deletion analysis of the promoter and evidence for cell-specific expression.

1986 ◽  
Vol 6 (2) ◽  
pp. 347-354 ◽  
Author(s):  
A Schmidt ◽  
P Rossi ◽  
B de Crombrugghe
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Chimeric Gene ◽  
Collagen Gene ◽  
3T3 Cells ◽  
Base Pairs ◽  
Specific Expression ◽  
Nih 3T3 ◽  
Cat Gene ◽  
Nih 3T3 Cells

A chimeric gene was constructed in which sequences between 2,000 base pairs upstream of the start of transcription of the mouse alpha 2(I) collagen gene and 54 base pairs downstream of this site were fused to the chloramphenicol acetyltransferase (CAT) gene. We present evidence suggesting that this collagen gene segment is sufficient for cell-specific expression of the chimeric gene. Indeed, the levels of CAT activity in transient expression experiments were at least 10 times higher after transfection of NIH 3T3 cells than after transfection of a mouse myeloma cell line, whereas much less difference was found after transfection of these two cell types with pSV2-CAT, a plasmid in which the early simian virus 40 promoter is fused to the CAT gene. Several deletions were introduced in the same 5'-flanking segment of the alpha 2(I) collagen gene, and the effects of these deletions were examined after DNA transfection of the chimeric collagen-CAT gene into NIH 3T3 cells. At least two segments broadly located between -979 and -502 and between -346 and -104 are needed for optimal expression of the chimeric gene. These results were obtained both in transient expression experiments and by analysis of pools of NIH 3T3 cells that were stably transfected with the different mutants. In general, the effects of the deletions on the activity of the alpha 2(I) collagen promoter were analogous, whether the plasmids harbored the simian virus 40 enhancer sequence or not, although the overall levels of expression of the chimeric gene were increased when the recombinant plasmids contained this sequence.

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