No abstract

We have identified an adenovirus type 5 (Ad5) early gene function located in early region 1 which is require for the production of early cytoplasmic mRNAs corresponding to early regions 2, 3, and 4. Mutant d1312 (lacks the segment between 1.5 and 4. Recently, we isolated a group of deletion mutants that lack portions of early region 1 (unpublished data). These mutants were isolated by selecting viral DNAs that lack the Xba I restriction endonuclease cleavage site-at 4 map units by the procedure of Jones and Shenk (7). These mutants are propagated in 293 cells (an Ad5-transformed human embryonic kidney cell line; ref. 8); they do not replicate in HeLa cells.In this report we show that the deletions in these mutants make predictable alterations in the cytoplasmic mRNAs encoded by early region 1. Further, we find that one of these mutants, d1312, defines a region 1 gene function that is required for the production of early cytoplasmic mRNAs corresponding to early regions 2, 3, and 4.MATERIALS AND METHODS Cells and Viruses. The 293 cells were provided by F. Graham and have been described (8 11g. RNA was isolated from cells (treated with 20,ug of cytosine arabinoside per ml) 8 hr after infection at a multiplicity of 50 plaque-forming units (PFU)/cell. Infected cells were divided into nuclear and cytoplasmic fractions. Cells were suspended in isotonic buffer (10 mM Tris1HCl, pH 7.8/1.5 mM MgCl2/150 mM NaCI), and Nonidet P-40 was added to 0.6% after the mixture was cooled to 40C. The mixture was held on ice for 10 mi. then mixed on a Vortex for 10 sec; the nuclei were pelleted by centrifugation. The supernatant was the cytoplasmic fraction and the pellet was the nuclear fraction after two additional washes in isotonic buffer.We prepared nuclear RNA by dissolving the nuclear pellet in a low pH sodium dodecyl sulfate buffer (50 mM NaOAc, pH 5.2/100 mM NaCI/10 mM EDTA/0.5% sodium dodecyl sulfate), extracting the RNA twice at room temperature with an equal volume of phenol (equilibrated with 50 mM NaOAc, pH 5.2), extracting it once with chloroform/isoamyl alcohol, 49:1 (vol/vol), and precipitating the RNA by addition of 2 vol of ethanol. The RNA was resuspended in 10 mM Tris-HCl, pH 7.4/10 mM MgCI2, electrophoretically purified DNase I was added to 50 ,ug/ml, and the solution was incubated at 37°C for 30 min. Finally, the RNA was reextracted with phenol and with chloroform/isoamyl alcohol, and precipitated with ethanol. Cytoplasmic RNA was prepared by mixing the cytoplasmic cellular fraction with 3 vol of 100 mM Tris-HCl (pH 9), extracting twice at room temperature with phenol (pH 9)/chloroform/isoamyl alcohol, 500:100:1 (vol/vol)

Our knowledge of the mechanisms that regulate transcription in higher eukaryotic cells has increased enormously during the past 2 years. Earlier studies, using a combination of in vitro mutagenesis and DNA-mediated gene transfer, identified two distinct types of cis-acting regulatory sequences: promoters, which are located close to the initiation site and act in a position-dependent fashion, and enhancers, which can be located far from the initiation site and act in a position-and orientation-independent fashion. Promoters can be subdivided into proximal elements, including the cap site itself and the TATA box, which is involved in fixing the site of initiation, and distal elements, which can be spread over several hundred base pairs. It is now clear that many promoters, particularly those of 'housekeeping' genes, lack TATA boxes and are instead composed of GC-rich elements that are often located within methylation-free islands (Bird 1986). Transcription controlled by this latter class of promoters often initiates at multiple sites. Many enhancer elements, for example, those of the immunoglobulin, insulin, and elastase genes, impose tissue-specific expression on adjacent promoters.These cis-acting elements operate by interacting with protein factors, many of which have now been identified by gel retardation and footprinting assays and some of which have been purified to homogeneity. In a few cases the corresponding genes have been cloned. In many of these studies well-characterized cis-acting elements of viruses, particularly of the DNA tumor viruses, have played a major role. At the recent ICRF-sponsored meeting on the Molecular Biology of SV40, Polyoma, and Adenoviruses (held in Cambridge, England, July 27-August 1, 1987) there was a major emphasis on such trans-acting factors. The results reported both identified new factors and considerably clarified the relationships amongst previously described factors.In this review we will discuss data reported at the meeting and, where appropriate, refer to recently published work. We will concentrate almost exclusively on work with DNA tumor viruses, referring to other systems only when there are direct connections to our major topic. Table 1 lists the factors of relevance, giving the DNA sequences they recognize, their synonyms, and the regulatory systems in which they are known to be involved.

We have investigated the E1A-inducible E3 promoter of adenovirus type 5 with respect to its ability to bind specific nuclear proteins. Four distinct nucleoprotein-binding sites were detected, located between positions -7 to -33, -44 to -68, -81 to -103, and -154 to -183, relative to the E3 cap site. These sites contain sequences previously shown to be functionally important for efficient E3 transcription. No major qualitative or quantitative differences were found in the binding pattern between nucleoprotein extracts prepared from uninfected or adenovirus-infected HeLa cells. Competition experiments suggest that the factors binding to the -154 to -183 and -81 to -103 sites are the previously identified nucleoproteins, NFI and AP1, respectively. The factor binding to the -44 to -68 site, which we term ATF, also interacts with other E1A-inducible promoters and is very similar and probably identical to the factor that binds to the cAMP-responsive element of somatostatin. We have purified this factor, which is a protein of 43 kD in size.

The close association of human papillomavirus type 16 DNA with a majority of cervical carcinomas implies some role for the virus in this type of cancer. To define the transforming properties of HPV-16 DNA in vitro we have now performed transfection experiments on baby rat kidney cells using HPV-16 DNA in conjunction with an activated ras gene. We have demonstrated that a 6.6-kb DNA fragment, containing the early genes of HPV-16 under the control of Moloney murine leukaemia virus long terminal repeats (MoMuLV-LTRs), cooperates with EJ-ras in transforming these cells. Both DNAs are required and neither alone is effective. The cooperating activity appears to reside in a protein or proteins derived from the E6/E7 region of the HPV-16 genome.