The infectious units in native and alkalidenatured preparations of DNA of herpes simplex virus were characterized with respect to their sensitivity to Neurospora crassa endonuclease, their sedimentation properties in high-salt, neutral sucrose gradients, and their sensitivity to hydrodynamic shearing forces. Infectious molecules in native preparations were resistant to N. crassa endonuclease, sedimented at 56 S, and were highly sensitive to shearing forces. After alkaline denaturation, infectious molecules became sensitive to the N. crassa enzyme, sedimented at 200 S, and were relatively resistant to shear. We conclude that both intact duplex molecules (z100 X 106 daltons) and intact single strands (-50 X 106 daltons) are capable of initiating productive infection.The chromosome of herpes simplex virus (HSV) is a linear, double-stranded DNA molecule of~100 X 106 daltons (1-4). A preliminary report (5) presented evidence that HSV DNA, isolated by mild extraction procedures, could infect primary rabbit-kidney cells or rabbit-skin fibroblasts in the presence of diethylaminoethyl-dextran. The infectious unit was shown to be DNase-sensitive, resistant to anti-HSV serum, and to have a density in CsCl of 1.7 g/cm3. Additional studies (Sheldrick and Laithier, in preparation) have shown that infectivity is protease-resistant, and that progeny virus issuing from DNA-infected cells carry genetic markers present in the infecting DNA. In this communication, we describe experiments that further characterize the infectious unit in native preparations as a molecule of HSV DNA. Moreover, we present evidence that intact single strands (-50 X 106 daltons) of HSV DNA are also infectious. MATERIALS AND METHODSCulture Medium. Eagle's minimal essential medium (Eurobio, Paris) was supplemented with 3.2 g of glucose, 2.7 g of tryptose phosphate (Difco), and 2.0 g of NaHCO3 per liter. All incubations using this medium were performed in an atmosphere of 5% CO2.Cells. The strain of rabbit-skin fibroblasts, RS537, used in this study was isolated and kindly provided by Dr. G. Orth (6). The cells were propagated at 370 in medium supplemented with 10% calf serum in the absence of antibiotics. Penicillin (100 units/ml) and streptomycin (100 yg/ml) were added to Abbreviations: HSV, herpes simplex virus; PFU, plaque-forming units; PBS, phosphate-buffered saline (pH 7.0). 3621 the medium for the growth of virus and for plaque assays. Cultures were trypsinized twice weekly and reseeded at l/4-1/5 the original cell concentration. The experiments described here were performed with cells between their 40th and 70th passages.Virus. The strain of HSV (subtype 1) used here is designated A44 and has been described (7). It produces a syncytial cytopathic effect in RS537 cultures and, in this sense, is similar to the MP variant described by Hoggan and Roizman (8).Virus Growth Qnd Purification. Confluent cultures of RS537 cells in 1-liter Roux bottles (about 2 to 3 X 107 cells per bottle)were infected with 1 to 2 X 107 plaque-forming units (PFU) of virus i...
The two-hybrid system was used to isolate cDNA clones encoding polypeptides that interact with the N-terminal region (activation domains A, B and C) of the Sp1 transcription factor. Among the 65 collected clones, 43 contained cDNA fragments with open reading frames. They corresponded to 13 genes encoding proteins of known function and to 15 genes, the proteins of which have no known function. Six overlapping cDNA clones corresponded to the Hsc70 protein. Host cell factor (HCF-1) and the KIAA0461 gene (encoding a putative Zn-finger protein of unknown function) were both identified through the isolation of three overlapping cDNA clones. Two cDNA fragments encoding the same region of the SREBP-2 transcription factor were independently selected and two overlapping cDNA clones corresponded to the splicing factor SF3A120. Two different cDNA clones encoded the N- and C-terminal region of the Oct-1 transcription factor. Transcription factors Elf-1 and TIEG, as well as HSph2, the putative human homologue of a murine polyhomeotic gene, were each represented by a single clone. Noticeably, for the four identified transcription factors, the DNA-binding domain was excluded from the selected polypeptides. In vitro binding of the selected polypeptides to the Sp1 protein was demonstrated for the four transcription factors and for the SF3A120, Hsc70, HCF-1, HSph2 and pKIAA0461(245) proteins. Four other cDNA clones encoding polypeptides of unknown function were tested in the in vitro binding assay. All four polypeptides were found to interact with Sp1 in this assay.
Clones of cells tumorigenic or nontumorigenic in nude mice have been previously isolated from the SW613-S human colon carcinoma cell line. We have already reported that tumorigenic cells overexpress the cytokeratin 18 (K18) gene in comparison with nontumorigenic cells and that this difference is mainly due to a transcriptional regulation. We now report that a 2,532-bp cloned human K18 gene promoter drives the differential expression of a reporter gene in a transient assay. A 62-bp minimal K18 promoter (TATA box and initiation site) has a low but differential activity. Analysis of deletion and substitution mutants as well as hybrid SV40-K18 promoters and reconstructed K18 promoters indicated that an important element for the activity of the K18 promoter is a high-affinity binding site for transcription factor Sp1 located just upstream of the TATA box. This Sp1 binding element, as well as the intron 1 enhancer element, stimulates the basal activity of the minimal promoter through mechanisms that maintain the differential activity. Gel shift assays and the use of an anti-Sp1 antibody have shown that both tumorigenic and nontumorigenic SW613-S cells contain three factors able to bind to the Sp1 binding element site and that one of them is Sp1. A hybrid GAL4-Sp1 protein transactivated to comparable extents in tumorigenic and nontumorigenic cells a reconstructed K18 promoter containing GAL4 binding sites and therefore without altering its differential behavior. These results indicate that the Sp1 transcription factor is involved in the overexpression of the K18 gene in tumorigenic SW613-S cells through its interaction with a component of the basal transcription machinery.
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