Promoter dependence of enhancer activity.

The interaction of enhancers with different promoters was studied by measuring the influence of two enhancers (from simian virus 40 and from Harvey sarcoma virus) on the activity of expression vectors that are identical except for their promoter region. The promoters examined were from the simian virus 40 early region, with or without its own 72-base-pair repeat, and the mouse beta major-globin gene. It is clear that the promoter acted upon strongly influences the level of activity of an enhancer.     

The expression of integrated plasmid DNA depends on copy number

The effect of copy number, integration site, and enhancers on the expression of stably integrated exogenous DNA was examined in Chinese hamster cells. Three similar plasmids were constructed with the mouse beta maj-globin promoter fused to the galK gene either with no enhancer or with the SV40 or Harvey sarcoma virus (HaSV) enhancer. Eighteen stable cell lines were obtained and characterized with respect to plasmid copy number and galactokinase activity. At copy numbers of four or less, the enhancers showed detectable activity and a DNase I hypersensitive site was present. Above four copies, gene activity decreased as the copy number increased, the enhancer sequences were apparently inactive, and the DNase I hypersensitive site disappeared. These data suggest that, at least in this model system, when exogenous DNA is integrated as multiple head-to-tail copies, the entire multigene unit expresses poorly and inappropriately. When the same exogenous DNA integrates as a single (or low number) copy, expression appears to be relatively normal as judged by enhancer stimulation and DNase I hypersensitivity.     

Correlation of gene expression and transformation frequency with the presence of an enhancing sequence in the transforming DNA.

The transformation frequency of cultured mammalian cells is increased 10- to 100-fold when certain DNA sequences are present in the transforming DNA. We wanted to determine whether enhancers, which stimulate gene expression, can cause this phenomenon. Three plasmids, each containing a galactokinase K (galK) gene, were used to transform galK- Chinese hamster cells. One plasmid has no enhancer, another has the simian virus 40 (72-base-pair repeat) enhancer, and the third has the Harvey sarcoma virus (73-base-pair repeat) enhancer. The presence of either enhancer significantly increased the appearance of GalK+ colonies. Galactokinase transient assays in this Chinese hamster strain in the presence of the same plasmids demonstrated an increase in GalK enzyme levels when either enhancer was present. These data indicate that there is a strong correlation between galK expression and transformation frequency that is dependent on the presence of an enhancer in the transforming DNA.     

Characterization of enhancer elements in the long terminal repeat of Moloney murine sarcoma virus

A series of recombinant molecules were constructed which direct the expression of the easily assayed gene chloramphenicol acetyltransferase. We have used these recombinants to show that the 73/72-base-pair tandem repeat unit from the Moloney murine sarcoma virus long terminal repeat shares a number of properties with the prototypic enhancer element, the simian virus 40 72-base-pair repeat. Specifically, the Moloney murine sarcoma virus sequence significantly enhances the level of gene expression at both 5' and 3' locations and in either orientation relative to the test gene. It is able to enhance gene activity both from its own promoter and from a heterologous (simian virus 40) promoter. The 73/72-base-pair subunits of the Moloney murine sarcoma virus enhancer differ in sequence by four nucleotides and also in the strength of their enhancer function. The promoter distal A repeat is at least three times as active as the promoter proximal B repeat in enhancing chloramphenicol acetyltransferase expression. Results of these studies also show that the enhancer sequence alone is unable to induce gene activity but requires other promoter elements, including a proximal GC-rich sequence and the Goldberg-Hogness box.     

Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.     

Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus long terminal repeat.

We have constructed a recombinant simian virus 40 (SV40) DNA containing a copy of the Harvey murine sarcoma virus long terminal repeat (LTR). This recombinant viral DNA was converted into an infectious SV40 virus particle and subsequently infected into NIH 3T3 cells (either uninfected or previously infected with Moloney leukemia virus). We found that this hybrid virus, SVLTR1, transforms cells with 10 to 20 times the efficiency of SV40 wild type. Southern blot analysis of these transformed cell genomic DNAs revealed that simple integration of the viral DNA within the retrovirus LTR cannot account for the enhanced transformation of the recombinant virus. A restriction fragment derived from the SVLTR-1 virus which contains an intact LTR was readily identified in a majority of the transformed cell DNAs. These results suggest that the LTR fragment which contains the attachment sites and flanking sequences for the proviral DNA duplex may be insufficient by itself to facilitate correct retrovirus integration and that some other functional element of the LTR is responsible for the increased transformation potential of this virus. We have found that a complete copy of the Harvey murine sarcoma virus LTR linked to well-defined structural genes lacking their own promoters (SV40 early region, thymidine kinase, and G418 resistance) can be effectively used to promote marker gene expression. To determine which element of the LTR served to enhance the biological activity of the recombinant virus described above, we deleted DNA sequences essential for promoter activity within the LTR. SV40 virus stocks reconstructed with this mutated copy of the Harvey murine sarcoma virus LTR still transform mouse cells at an enhanced frequency. We speculate that when the LTR is placed more than 1.5 kilobases from the SV40 early promoter, the cis-acting enhancer element within the LTR can increase the ability of the SV40 promoter to effectively operate when integrated in a murine chromosome. These data are discussed in terms of the apparent cell specificity of viral enhancer elements.     

Transcriptional trans-activating function of hepatitis B virus

The ability of hepatitis B virus (HBV) to stimulate the expression of a cellular gene was investigated by using a transient-expression system. A plasmid in which the expression of the bacterial chloramphenicol acetyltransferase (cat) gene had been placed under the control of the DNA sequences that regulate the expression of the human beta-interferon gene was constructed. In Vero cells, cotransfection of the 2.7-kilobase BglII DNA fragment of HBV together with the test plasmid containing the cat gene resulted in stimulation of the expression of the cat gene. This HBV DNA fragment was specific in its trans-activation; no significant stimulation of CAT activity was observed in constructs when the promoter and enhancer elements were derived from the murine sarcoma viral long terminal repeat, Rous sarcoma virus, BK virus, or simian virus 40. Results of subcloning of the HBV DNA fragment indicate that the trans-activating function resides in a 944-base-pair EcoRV-BglII DNA fragment of the HBV genome that contains the X structural gene and its promoter element. Removal of the promoter from the X structural gene resulted in loss of the trans-activating function. A frameshift mutation within the X gene region also eliminated the trans-activating activity. These results suggest that the X antigen could play a role in HBV infections by activating the expression of cellular genes.     

Negative transcriptional regulatory element that functions in embryonal carcinoma cells.

We have cloned the polyomavirus mutant fPyF9, which persists in an episomal state in F9 embryonal carcinoma cells (K. Ariizumi and H. Ariga, Mol. Cell. Biol. 6:3920-3927, 1986). fPyF9 carries three copies of exogenous sequences, the prototype of which is a 21-base-pair repeat (box DNA), in the region of the enhancer B domain of wild-type polyomavirus DNA. The consensus sequence, GCATTCCATTGTT, is 13 base pairs long. The box DNA inserted into fPyF9 appeared to come from a cellular sequence and was present in many kinds of DNAs, including F9 chromosomal DNA. The biological function of box DNA was analyzed by chloramphenicol acetyltransferase expression assays, using chimeric plasmids containing box DNA conjugated with simian virus 40 promoter elements. The results showed that box DNA repressed the activities both of the simian virus 40 promoter and enhancer only in transfected undifferentiated F9 cells and not in differentiated LTK- cells. Box DNA functioned independently of orientation and position with respect to the promoter in an enhancerlike manner, although the effect of box DNA was opposite that of the enhancer. The XhoI linker insertion into the consensus sequences of box DNA abolished the repression activity, and the protein(s) recognizing the consensus sequences was identified only in F9 cells, not in L cells. These analyses suggest that box DNA may be a negative regulatory element that functions in undifferentiated cells.     

Host-specificities of papillomavirus, Moloney murine sarcoma virus and simian virus 40 enhancer sequences.

The bovine papillomavirus (BPV-1), Moloney murine sarcoma virus (MoMuSV) and simian virus 40(SV40) genomes have been shown to contain sequences termed 'enhancers' which activate the expression of linked genes. DNA fragments containing these three enhancers have been inserted into recombinant plasmids upstream from the herpes simplex virus thymidine kinase (tk) gene, and their effect on tk expression monitored. Two types of assay have been used. Firstly, the ability of recombinant plasmids to transform TK- recipient cells to a TK+ phenotype was measured. Secondly, the amount of tk-specific RNA and TK enzyme activity transiently expressed after DNA transfection was determined. Both types of assay gave similar results. The enhancers increased tk gene expression by regulating the amount of full length tk mRNA present shortly after transfection independent of gene copy number. Furthermore, marked species specificity in the relative efficiencies of different enhancers was observed, including that of the BPV-1 enhancer for the first time. The MoMuSV enhancer showed preference for murine fibroblasts, while the papillomavirus enhancer showed a marked preference for bovine cells. In contrast, the SV40 enhancer gave the same relative increase in tk gene expression in the murine, rat, bovine and human cells tested.     

Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5

We examined the promoter activity of the 1.3-kb chicken beta-actin gene sequence located between the 5' flanking region and the proximal region of the second exon. This promoter region showed higher promoter activity than the simian virus 40 (SV40) early promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) as assayed by transient lacZ gene expression in mouse L cells. Furthermore, replacement of the 3' splice sequence in this promoter by that derived from the rabbit beta-globin gene resulted in a approximately 2.5-fold enhancement in the synthesis of beta-galactosidase (beta Gal). Introduction of the SV40 origin of DNA replication (ori) into the vector carrying this hybrid promoter, which we designate the AG promoter, markedly enhanced the production of beta Gal in an SV40 T antigen-producing cell, BMT10. We have constructed a useful vector containing the strong AG promoter, several unique restriction sites, a SV40 polyadenylation signal and the SV40 ori for transient expression of cDNA in BMT10 or COS cells. We demonstrate the use of this vector for efficient production of interleukin-5 in BMT10 cells.