Differential activation of the mouse beta-globin promoter by enhancers.

Abstract

A series of plasmids was constructed to study the effect of two enhancers, the simian virus 40 72-base-pair repeat and the Harvey sarcoma virus 73-base-pair repeat, on the mouse ma'J-globin promoter. These plasmids contain the mouse Pm"-globin promoter linked to the Escherichia coli galK gene, thus allowing galactokinase enzyme activity to be used as a measure of promoter function. In CV-1 (primate) cells, it was found that an enhancer is required for optimal promoter activity and that the simian virus 40 (primate) enhancer increases galactokinase fourfold more than the Harvey sarcoma virus (mouse) enhancer. In L (mouse) cells, however, the Harvey sarcoma virus enhancer is 1.3-fold stronger than the simian virus 40 enhancer. These data support the hypothesis that enhancer activity can be species specific. Furthermore, when both enhancers are present on the same plasmid, their effect is additive on the ,-globin promoter whether the plasmid is in CV-1 cells or L cells.The mouse ,B-globin gene serves as a valuable model for the study of gene expression in mammals. We wished to develop expression vectors which would allow us to optimize the expression of cloned mouse 3-globin genes that are transferred into mouse tissue culture cells or into intact animals. Important elements that have been shown to stimulate the expression of certain mammalian genes have recently been discovered. These are cis DNA sequences called enhancers (1,2,(4)(5)(6)(7)(11)(12)(13)(14). As a first step toward obtaining regulated globin gene expression in mice, we examined the effect of two different enhancing sequences, the monkey simian virus 40 (SV40) 72-base-pair (bp) repeat and the mouse Harvey sarcoma virus (HaSV) 73-bp repeat, on the expression of a cloned mouse P'ai-globin promoter in both monkey (CV-1) cells and mouse (L) cells.Specifically, we asked several questions. (i) Studies have been performed with the SV40 enhancer with its natural promoter, the SV40 early promoter (2,4,6,11,13). Does the SV40 enhancer have a similar effect on the mouse pglobin promoter? (ii) The SV40 and the murine Moloney sarcoma enhancer act differently on the SV40 early promoter (11), and the SV40 and polyoma enhancers show cell-specific stimulation of the SV40 and rabbit P-globin promoters (4). Is this cell (species)-specific effect a general one? (iii) Do two different enhancers have an additive effect on promoter activity?To answer these questions, we constructed a series of plasmids containing the mouse jmajglobin promoter linked to the Escherichia coli galactokinase gene (galK). This allows us to measure galactokinase (Gal K) levels as a function of the activity of the P-globin promoter.Some constructs also contain a second assayable E. coli gene, gpt, which is driven by the SV40 early promoter, giving an internal control for transformation frequency. These constructions are based on the system described by Schumperli et al. (16). The SV40 72-bp repeat and the HaSV 73-bp repeat were added singly and in combination to these constructs. The Gal K and gpt...

Structure of the galactokinase gene ofEscherichia coli, the last (?) gene of thegaloperon

“…6 for a review). The use of galK as a selective marker has also been adapted to develop gene fusion vectors to study transcriptional regulatory elements in the gram-positive bacteria Streptomyces (7), yeast (8)(9)(10)(11) and higher cell systems (5,(12)(13)(14)(15).…”

Functional Analysis of the Transcription Control Region Located Within the Avian Retroviral Long Terminal Repeat:

“…Although certain promoters show detectable transcriptional activity in the absence of a detectable enhancer element, activation of a promoter by an enhancer appears necessary for high levels of transcription to occur. Indeed, some promoters are essentially inactive in the absence of an enhancer element (4,44).…”

“…Enhancers can show both host and tissue specificity and can confer this specificity on the interacting promoter element (1,4,15,26,47). Although the eucaryotic TCR can be divided into the three functionally distinct elements described above, the relative importance of these elements in the control of gene * Corresponding author.…”

“…To directly test this hypothesis, we used an assay which measures the ability of DNA fragments, in this case LTRs, to activate the mouse ,-globin gene promoter when located in cis. The P-globin promoter requires activation by an enhancer element to function, and the level of 3-globin promoter activity then observed is a measure of the efficiency of the enhancer being tested (4). In the parent plasmid pPB12, the mouse P-globin promoter controls the expression of the E. coli galactokinase (galK) gene (4).…”

“…The P-globin promoter requires activation by an enhancer element to function, and the level of 3-globin promoter activity then observed is a measure of the efficiency of the enhancer being tested (4). In the parent plasmid pPB12, the mouse P-globin promoter controls the expression of the E. coli galactokinase (galK) gene (4). The pKR2 clones used contained different parts of the SR-Btd LTR, as indicated in Table 1, inserted into the same site in pPB12, upstream of the P-globin promoter in the opposite transcriptional orientation.…”