Activation of mutated simian virus 40 enhancers by amplification of wild-type enhancer elements.

We show that duplication of any one of three separate simian virus 40 enhancer elements, A, B, or C, can compensate for loss of function in the remaining two. Simian virus 40 revertants containing point mutations within the A and C (dpm16) or B and C (dpm26) enhancer elements contain tandem duplications that include the remaining wild-type element. These simple tandem duplications can create enhancers 25-fold more active than that of the parental mutant. These revertants can arise by illegitimate recombination between heterologous viral genomes. This was demonstrated by the recombinants resulting from a mixed infection with the viruses dpm16 and dpm2, which contain mutations in the A and C elements and the B element, respectively.     

The HeLa cell protein TEF-1 binds specifically and cooperatively to two SV40 enhancer motifs of unrelated sequence

We have purified a protein (TEF-1) that specifically binds to two sequence unrelated motifs (GT-IIC and Sph) of the simian virus 40 (SV40) enhancer. TEF-1 binds cooperatively to templates containing tandem but not inverted or spaced repeats of its cognate motifs. This cooperative binding correlates with the ability of the tandem repeats to generate enhancer activity in vivo. In contrast, TEF-1 and a second SV40 enhancer binding protein, TEF-2, bind independently to templates containing the cognate motifs of both proteins (GT-I and either GT-IIC or Sph motifs) even though these motifs cooperate in enhancer activity in vivo. These results allow us to distinguish different classes of enhancer factors.     

Three individual regulatory elements of the promoter positively activate the transcription of the murine gene encoding granulocyte colony-stimulating factor.

At least three regulatory elements GPE1, GPE2 and GPE3 (G-CSF promoter elements) controlling the gene (G-CSF) encoding granulocyte colony-stimulating factor (G-CSF) are indispensable for the constitutive expression of the G-CSF gene in human CHU-2 cells and for its lipopolysaccharide(LPS)-inducible expression in macrophages. The enhancer activities of each regulatory element were examined with or without the SV40 enhancer element placed downstream from the reporter gene. A GPE1 tetramer mediated the constitutive expression in CHU-2 cells, and the LPS-inducible expression in macrophage cell lines, while the GPE2 element was active in CHU-2 and LPS-treated macrophage cell lines only in combination with the SV40 enhancer. A GPE3 tetramer had efficient enhancer activity in CHU-2 cells but not in macrophage cell lines without the SV40 enhancer. In combination with the SV40 enhancer, GPE3 worked as an LPS-inducible enhancer element in macrophage BAM3 cells. Gel retardation assay indicated that the CHU-2 and the macrophage cells contained nuclear factors which specifically bound to each GPE sequence.     

Minimal transcriptional enhancer of simian virus 40 is a 74-base-pair sequence that has interacting domains.

We have assayed the ability of segments of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer region to activate gene expression under the control of the SV40 early promoter and to compete for trans-acting enhancer-binding factors of limited availability in vivo in monkey CV-1 or human HeLa cells. The bacterial chloramphenicol acetyltransferase and the herpes simplex virus type 1 thymidine kinase genes were used as reporters in these assays. A 94-bp sequence located between SV40 nucleotides 179 and 272, including one copy of the 72-bp repeat, has been termed the minimal enhancer in previous studies. In the present study, we found that the 20-bp origin-proximal region located between nucleotides 179 and 198 was dispensable, since its removal caused only a slight reduction in enhancer activity. However, the deletion of another 4 bp up to nucleotide 202 abolished the enhancer activity. We propose that the minimal enhancer is a 74-bp sequence located between nucleotides 199 and 272, including 52 bp of one copy of the 72-bp repeat and a 22-bp adjacent sequence up to the PvuII site at 272. The nonamer 5'-AAGT/CATGCA-3', which we term the K core, occurred as a tandem duplication around the SphI site at nucleotide 200, and we found that this duplication was essential for enhancement and factor-binding activities. A heterologous core element (which we term the C core), 5'-GTGGA/TA/TA/TG-3', identified earlier (G. Khoury and P. Gruss, Cell 33:313-314, 1983; Weiher et al., Science 219:626-631, 1983) also occurred in duplicate, with one of the copies located within the 22-bp sequence near nucleotide 272 present outside the 72-bp repeat. We provide direct evidence that this 22-bp sequence augments enhancer activity considerably. We also found that in addition to the heterologous interaction occurring normally between the K and C cores within the minimal enhancer, certain homologous interactions were also permitted provided there was proper spacing between the elements.     

Comparison of CMV, RSV, SV40 viral and Vlambda1 cellular promoters in B and T lymphoid and non-lymphoid cell lines.

Determining the activity of viral and cellular regulatory elements in B or T lymphoid cell lines would facilitate appropriate utilization of the regulatory sequences for gene transfer- and expression-dependent applications. We have compared the activity of the CMV, RSV and SV40 viral promoter/enhancers as well as the Vlambda1 cellular promoter, in three B cell lines (REH, SMS-SB, C3P), three T cell lines (CEM, Jurkat, ST-F10), and two non-lymphoid cell lines (K-562, HeLa) using the luciferase reporter gene. In B cell lines, the activity of the CMV promoter/enhancer construct was the highest ranging from 10- to 113-fold greater than that of SV40. In contrast, in T cell lines the RSV promoter/enhancer activity was 11-65-fold higher than that of SV40. The Vlambda1 promoter activity was close to that of SV40 promoter/enhancer in most of the cell lines tested. We conclude that CMV and RSV promoter/enhancers contain stronger regulatory elements than do the SV40 and Vlambda1 for expression of genes in lymphoid cell lines.