The effect of the human cytomegalovirus immediate early region 1 enhancer on transcription was studied in vitro with HeLa cell nuclear extract. Stimulation of in vitro transcription mediated by the enhancer element involves its recognition by specific trans-acting factors present in the nuclear extract. DNase I protection analysis was used to determine at the nucleotide level those enhancer sequences that interact with nuclear factors. At least nine sites of protein-DNA interaction were detected over -400 base pairs of enhancer sequence. The regions of nuclease protection are associated with 21-, 19-, 18-, and 17-base-pair repeat elements as well as with a unique sequence, creating a large nucleoprotein complex. The relationship between the protein binding and the activity of the immediate early region 1 enhancer is discussed.Human cytomegalovirus (HCMV), a member of the herpesvirus family, is the etiologic agent of a wide spectrum of human diseases (1, 2). Upon infection, the HCMV genes are expressed in three sequential phases MATERIALS AND METHODS Preparation of DNA. The recombinant plasmids pPSCAT, pSSCAT and pCMV(-524)CAT, pCMV(-65)CAT were obtained from J. Nelson and R. Ruger, respectively. These constructs contain various 5'-flanking sequences of the HCMV (strain AD169) IEJ gene linked to the bacterial chloramphenicol acetyltransferase gene (see Fig. 1A). Plasmids pE1, pE2, and pE3 contain BAL-31 deletion fragments (prepared by R. Ruger) overlapping the enhancer and promoter region of the IEJ gene that were cloned into the HindIII and BamHI sites of pTZl8R (see Fig. 1A). The competitor fragments (El, E2, and E3) were gel-purified from their respective plasmids after cleavage with EcoRd and HindIII restriction enzymes.Nuclear Extract Preparation and Fractionation. Nuclear extract from HeLa cells was prepared as described by Dignam et al. (20). The fractionation of the nuclear extract on columns of heparin-agarose (Bio-Rad) and DEAE-Sepharose CL-6B (Pharmacia) was performed according to the procedure of Dynan and Tjian (21). The heparin-agarose and DEAE-Sepharose columns were step-washed with 0.4 M and 0.225 M KCl, respectively, The flow-through fractions (HAO.1, DEO.1) and the step-washed fractions (HAO.4, DEO.225) from the column chromatography were used in the DNase I protection analysis.In Vitro Transcription Assays. RESULTSThe HCMV IEJ Gene Promoter Is Transcriptionally Active in Vitro. Run-off assays were performed to determine whether the crude nuclear extract prepared from the nuclei of uninfected HeLa cells contained the necessary factors for initiating and sustaining transcription from the HCMV IEl promoter. The DNA templates pPSCAT and pSSCAT, containing the IEl promoter and 5'-flanking region, were cleaved with the restriction endonucleases Pvu II or EcoRI, which truncate the template 165 or 260 base pairs downstream of the IEl cap site, respectively, generating run-off transcripts that could be visualized by electrophoresis. For all the templates tested, the size of the run-off transc...
Deficiency or abnormality of coagulation factor VIII (FVIII) causes a bleeding disorder called hemophilia A. Treatment involves FVIII concentrates prepared from pooled human plasma or recombinant FVIII (rFVIII) prepared from mammalian cell culture. The cost of highly purified FVIII or rFVIII is a major factor in hemophilia therapy and restricts prophylaxis. We have sought to generate a new source of rFVIII by targeting expression of the human FVIII cDNA to the mammary gland of transgenic pigs using the regulatory sequences of the mouse whey acidic protein gene. The identity of processed heterodimeric rFVIII was confirmed using specific antibodies, by thrombin digestion and activity assays. The secretion of as much as 2.7 micrograms/ml of rFVIII in milk was over tenfold higher than in normal plasma. Up to 0.62 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma.
In this paper we demonstrate that modulator sequences upstream of the enhancer of the major immediateearly promoter of human cytomegalovirus exert a differential effect on the level of transcription in a variety of cells and that this region has the capacity to interact with specific nuclear protein. Depending on the cell type, these modulator sequences increased or decreased transcriptional activation from the IEl gene promoterenhancer. The cell lines identified in this report should be useful to study the molecular mechanism of cell-specific transcriptional repression and activation exerted by the major immediate-early promoter upstream region.Although human cytomegalovirus (HCMV) nonproductively infects cells of many types from humans (8,16,17) and other species (2, 11), the virus replicates in vitro only in fibroblasts of human origin. Transcription of the first viral genes expressed, the immediate-early (IE) genes, are mainly dependent on host cell factors (1, 5-7, 20, 21), suggesting that this may constitute the primary event at which expression of the viral genome is regulated. Regulatory sequences of the HCMV major IE gene, the IEl gene, encompass a polymerase II promoter between +1 and -65 (6, 19); a strong and complex enhancer between -66 and -524 (1, 5, 7), which is active in many different cell types; a cluster of nuclear factor 1-binding sites between -600 and -720 (9, 10); and modulator sequences between -750 and -1145 (14, 15). These modulator sequences negatively regulate expression in nonpermissive undifferentiated teratocarcinoma (Tera-2) cells but positively influence expression in permissive differentiated cells (14,15). In the present study we have addressed the question of whether the modulator region is functional in cell types other than the embryonal carcinoma cell system and whether indeed it may contribute to cellspecific expression.Recombinant plasmids containing the modulator (A1145) or with the modulator sequences truncated (A525), which were previously used to define the modulator region (15) A1145 and A525 in cells of various lineages were measured by scintillation counting of conversion rates from chloramphenicol to its acetylated forms. All cells were cotransfected with pRSV-luciferase as a transfection control (3), and the results with A1145 and A525 were adjusted according to the relative luciferase activity in the cell extracts. The effect of the modulator sequences on CAT activity differed depending on cell type (Fig. 1). A twofold negative effect was observed in H9, U937, and SW480 cells, but expression in Jurkat, 293, Raji, and Namalwa cells was barely affected. In B cells the expression was augmented about three-to fourfold by the upstream sequences. These results clearly indicate that the HCMV modulator sequences can influence IE promoterenhancer activity in vivo in cell types other than teratocarcinoma cells. In particular, the cis-acting sequences negatively modulate expression in some T cells and rectal epithelial cells, in which HCMV is found during natural infection...
The possibility of DNA-binding proteins interacting in vitro with the polymerase H transcriptional machinery was explored by using a competition assay with individual target sequences for enhancer-binding factors. Transcription factors binding to at least five specific enhancer sequences mediate the activity of the human cytomegalovirus immediate-early 1 gene in vitro. Furthermore, our data suggest that individual DNA-bound enhancer factors can interact with the promoter transcription complex.The human cytomegalovirus (CMV) immediate-early 1 gene (IE1) enhancer is a strong potentiator of transcription both in vivo and in vitro (1, 2, 10). The sequence structure of the enhancer region, which extends from about nucleotide -65 to -530, exhibits a highly modular complexity. We have reported previously that five distinct sequence motifs within the enhancer region are targets for specific nuclear proteins (2). Here we present an approach for studying the involvement of these sequence-specific DNA-binding proteins with the transcriptional machinery. This involves the synthesis of oligonucleotides corresponding to specific protein-binding sites and a study of their effect in the in vitro transcription competition assay (4,8,9,12). An advantage of this approach is that it can be used to directly monitor the transcriptional machinery in a crude in vitro system that upon further fractionation might lose transcription factors, which may go undetected. Also, the assay avoids an alteration of the DNA sequence structure that may be important for enhancer activity. The assay also minimizes the possibility of competition for several proteins capable of interacting with larger restriction fragments (2,8,9,12).In pursuit of this approach, oligonucleotides corresponding to the target sequences ( Fig. 1A) were synthesized and prepared for the in vitro transcription competition assay. The in vitro transcription assay and the preparation of HeLa cell nuclear extract have been described previously (2). The templates, pCMV(-524)CAT and pCMV(-65)CAT, used for runoff analysis were a complete PvuII-HindIII restriction enzyme digest (2). The amount of template present in each in vitro synthesis was 300 ng per 25-,ul reaction. The nuclear extract (6 IlI), in 21 p.l of buffer containing 12 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.9), 10% (vol/vol) glycerol, 1.6 mM dithiothreitol, 0.12 mM EDTA, 60 mM KCl, 2 mM MgCl2, and 5 U of placental ribonuclease inhibitor, was initially incubated for 10 min at 4°C with or without the appropriate competitor DNA, as indicated. The template and nucleoside triphosphates were subsequently added, and the in vitro synthesis was allowed to proceed for 30 min at 30°C. The synthesized RNA was processed and analyzed on a 4% sequencing gel as described previously (2).Increasing the amount of several different nonspecific DNAs in the transcription reactions did not significantly alter the transcriptional activity of the enhancer-containing * Corresponding author. template, pCMV(-524)CAT (Fi...
Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys 2-Arg-1 and removal of a Lys'56-Arg'57 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.
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