Elements within the enhancer of T-lymphomagenic SL3-3 virus were examined for their contributions to transcriptional activity in T lymphocytes and non-T cells. A region containing two sequences homologous to the enhancer core consensus sequence and a sequence homologous to the binding site for factor LVb was found to have the largest effect on activity. Evidence was obtained that suggests that the activity of this region was greater in T lymphocytes than in non-T cells and that multiple elements within it were necessary for activity. A second region, containing sequences homologous to the binding site of factor NF-I and the glucocorticoid response element, had about a twofold effect on transcription in both T lymphocytes and non-T cell lines. The twofold effect was seen whether the region containing the cores and LVb site was present or not. These results indicate that the most important region for the specificity of SL3-3 enhancer activity and, presumably, for viral leukemogenicity comprises the core elements and the LVb site. DNA-protein-binding studies demonstrated that one cellular factor, S/A-CBF, bound to both core elements, while a second cellular factor, S-CBF, bound to only one of them. In combination with earlier studies, this indicates that cells contain multiple factors that bind to the critical region.

Transcriptional activities of the long terminal repeats (LTRs) of various murine leukemia viruses were tested in the cytotoxic T-cell lines CTLL-1 and CTLL-2. In contrast to T-lymphoma cells, in which the LTRs of T-lymphomagenic virus SL3-3 and Moloney murine leukemia virus are more active than those of other viruses, transcriptional activity in these mature, interleukin-2-dependent cells is not correlated with the specificity of viral leukemogenicity. Several approaches were used to investigate the molecular basis for LTR activity differences in lymphoma cells and mature cytotoxic T cells. Deletion analysis of the Moloney virus LTR showed that the direct repeats associated with enhancer activity have, at most, a slight effect on expression in CTLL-1 cells, whereas they stimulate expression six- to eightfold in T-lymphoma cells. This suggests that the mature T-cell line lacks one or more factors present in T-lymphoma cells that function to augment transcription from the Moloney murine leukemia virus LTR. We also used recombinant viral LTRs to investigate the role of the enhancer core element of SL3-3 in CTLL-1 and CTLL-2 cells. A one-base-pair difference between the core sequences of SL3-3 and nonleukemogenic Akv virus, which is important for SL3-3 activity in T-lymphoma cells, had no effect in these cells. The inability to distinguish the single-base-pair difference in expression assays was correlated with the absence of binding of a cellular factor, S-CBF, to the SL3-3 enhancer core in extracts of CTLL-1 and CTLL-2 nuclei. These studies may have implications for identification of the target cells for viral leukemogenesis, as well as for tracing of changes in the transcriptional machinery during T-lymphocyte differentiation.

Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins. Repair depends on the ability of cellular factors to distinguish the damaged sites. Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites.A binding assay based on the use of .3-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins. Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA. These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins.Cellular DNA is continually subjected to spontaneous and environmentally induced damage that, if left unrepaired, can result in cell death, mutation, or neoplastic transformation. Most DNA repair mechanisms, including base and nucleotide excision repair as well as direct reversal repair pathways, involve the participation of enzymes that recognize DNA structural alterations (1-3). An apurinic (AP) or apyrimidinic site is produced by the hydrolysis of the N-glycosylic bond that links a base to deoxyribose, and such sites are an important type of DNA damage. Spontaneous release of purines constitutes one of the most frequently occurring types of DNA damage under normal physiological conditions (4,5). AP and apyrimidinic sites are also generated by various chemical or radiation-induced modifications ofDNA bases or by the action of DNA repair glycosylases (1-3). They are mutagenic in bacterial and mammalian systems (6-9). Hence, an understanding ofthe proteins involved in the detection and repair of AP sites and of the genes encoding those proteins is important for comprehending the biological consequences of this type of damage.We report here the use of an electrophoretic assay to detect factors in mammalian cells that bind to DNA containing AP sites. We also report the development of an approach for the cloning of cDNAs encoding proteins that bind preferentially to DNA containing AP sites.MATERIALS AND METHODS DNA Probes. Restriction fragments to be used in the binding assays with mammalian nuclear extracts were isolated from various plasmids. They were 3'-end-labeled using the Klenow fragment of DNA polymerase I and [a-32P]dNTPs. AP sites were then introduced into the probes. Radiolabeled DNA was methylated by incubation in 200 ,ul of 50 mM sodium cacodylate/1 mM EDTA with 1 1.l of dimethyl sulfate (DMS) for 30 sec at room temperature (10, 11).Reactions were terminated by the addition of 50 gl of 1.5 M NaCl/1.0 M 2-mercaptoethanol containing poly(dI-dC)-poly-(dI-dC) (Pharmacia) at 100 gg/ml. Samples were then ethanol-precipitated, resuspended in 10 mM Tris-HCl, pH 4.5/1 mM EDTA, and incubated at 50'C for 30 min. Undamaged probes were treated identically except that DMS was...

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A conditional expression system was established whereby the human K-ras, v-src, and v-mos genes were cloned into a conditional expression vector downstream of the dexamethasone-inducible mouse mammary tumor virus long terminal repeat. Rat-1 fibroblasts were transfected with these constructs and selected in medium containing G418. Cloned transfectants were isolated and characterized for absolute dependence on dexamethasone for expression of oncogene products and anchorage-independent growth in soft agar. Expression of activated p21K-ras(val12) enabled the fibroblasts to degrade extracellular matrix collagen secreted by murine microvessel endothelial cells. Concurrent with p21K-ras(val12) induction a proteinase with the characteristic size and substrate specificity of transin, the murine homologue of the human matrix metalloproteinase stromelysin, was expressed and secreted. Induction of v-mos and v-src oncogenes resulted in little or no detectable transin expression respectively coinciding with a relative or absolute failure to increase degradation of extracellular matrix collagen. This study suggests that in this system the expression of the ras oncogene can contribute to the in vitro invasive behavior of tumor cells by upregulating the production of a metalloproteinase capable of degrading collagen synthesized by vascular endothelial cells.