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With standard protocols for DNA infection, only a small fraction (about 4%) of monkey cells exposed to purified DNA of simian virus 40 (SV40) exhibits signs of infection. We have devised a protocol by which we can extend the time of exposure of BSC-1 cells to DNA in the presence of low concentrations of DEAE-dextran. The efficiency of infection is proportional to the time ofexposure. With an 8-hr exposure, we are reproducibly able to infect 25% of the cells, and we have been able to achieve levels of infection as high as 50% with a 16-hr exposure. The percentage ofcells infected was measured either by scoring for nuclei positive for SV40 tumor antigen or by an infectious centers assay. We also report the use of ethidium bromide as a nonspecific nuclear counterstain in the immunofluorescence assay for SV40 tumor antigen.When a monolayer of monkey cells is infected at high multiplicity with particles of simian virus 40 (SV40), virtually all the cells become positive for SV40 tumor (T) antigen and exhibit cytopathic effects. In contrast, several investigators have found that when the same cells are incubated with high concentrations of SV40 DNA, even in the presence of a facilitator such as DEAE-dextran (1), only a small fraction (1-6%) of the cells exhibits signs of infection (2, 3). This low efficiency of DNA infection has frequently made interesting experiments arduous if not impossible. For example, it is difficult to study the biological properties of many potentially interesting viral mutants because one must begin with an inefficient DNA infection. Similarly, the inefficiency of DNA infection has limited the usefulness of SV40 DNA as a cloning vehicle in mammalian cells. For this reason, we decided to investigate ways to increase the efficiency of SV40 DNA infection.Two models have been proposed to explain the low efficiency of infection of viral DNA. In the first, a small subset of cells is thought to be competent to respond to a DNA infection because the cells are sensitive during a limited portion of the cell cycle (4). Because only a few cells in a randomly growing population would be in a susceptible phase ofthe cell cycle at any one time, this model predicts that the percentage of cells infected should be proportional to the time of exposure to DNA. In a second model, the fraction of cells that can be infected with DNA is thought to be controlled by "self-interference" in a way that would be independent of the cell cycle (2). This type of interference might occur, for example, because of competition between DNA molecules for a limited supply of cellular factors required for a successful infection. If limiting factors in the cell become available with time, one might predict that longer exposures of cells to DNA should increase the efficiency of infection.Both models suggest that the efficiency of infection with SV40 DNA could be increased by lengthening the period during which cells are exposed to DNA. To this end, we have devised a protocol in which monkey cells can be incubated for several h...

Polyoma virus mutants of four functionally distinct groups have been mapped by the marker rescue technique using restriction enzyme fragments of wild-type viral DNA. Nontransforming host-range mutants map in the proximal part of the early region of the viral genome. The (3, 4, *).Temperature-sensitive (ts) mutants fall into three complementation groups-an early group (ts-a) that is defective in the initiation of transformation, and two late groups that are unaffected in transformation (5). One. mutant, ts-3, fails to complement all other mutants, and is probably defective in a virion protein (6).Physiological properties of hr-t mutants set them clearly apart from all known temperature-sensitive mutant groups, but do not permit one to decide whether they are altered in an "early" or a "late" viral function. In productive infection they complement well with ts mutants of both early and late classes; they also complement with ts-a mutants for transformationtt.We have employed the physical mapping procedure, devised and applied to bacteriophage XX-174 by Hutchison and Egdell (7), in order to map hr-t and ts mutants of polyoma virus. This procedure has recently been applied to ts mutants of simian virus 40 (SV40) (8-10) and polyoma virus (11). Our results show that hr-t mutants map in the proximal part of the region of viral DNA that is transcribed early during productive infection and in transformed cells. Alterations within the same small segment

Differential mRNA display (DD) is potentially a powerful method for identifying genes that are over-or under-expressed in one cell type relative to another. In this technique, as first described by Liang and Pardee (1), total RNA is isolated from two cell types to be compared, and first strand copies of both RNAs are made by reverse tanscription, using an oligo-dT primer that has a specific dinucleotide at its 3' end (the 3' amplimer). This 3' amplimer and an arbitrarily-chosen lOmer (the 5' amplimer) are then used in the polymerase chain reaction (PCR) to amplify cDNAs to which the 3' and 5' amplimers both hybridize. A radioactive nucleotide is included in the PCR reactions so that the products can be run side-by-side on a sequencing gel and visualized by autoradiography. Bands that appear on the display ofRNA from one cell type but not the other correspond to differentially-expressed mRNAs. These bands can be excised from the gel and amplified with the same primers used for the original display, and the resulting PCR product can be used to probe Northern blots of polyA+ RNA to confirm the identification of a differentially-expressed mRNA.Although this technique has been used to identify differentiallyexpressed genes, two serious problems have limited its usefulness. First, the pattern of differential expression seen on the 'display' often cannot be reproduced on Northern blots (2,4). These false positives can arise with a frequency >70% (4). A second difficulty is that the cDNAs obtained represent only -300 bp at the extreme 3' end of the mRNA (usually in the 3' untranslated region). Since this part of the mRNA is frequently not included in GenBank and varies greatly from organism to organism, cDNAs identified by DD cannot always be matched with genes that have already been characterized. To obtain the translated region of the mRNA, experimenters have resorted to the time-consuming process of screening cDNA libraries (2,4), which also can be problematic, because the 3' untranslated regions of different genes can be quite similar.We have modified the original DD protocol to reduce the fiequency of false positives, and have devised a strategy that allows us to easily walk, in steps of -1 kb, along the mRNA toward its 5' end. Although we have used this walking technique only in connection with DD, our approach should be useful in other situations when access to sequences 5' to known mRNA sequences is desired.To reduce the number of false positive cDNAs that arise with DD, we use the following strategy. (i) We either a) display PCR products from two 'uninduced' cell lines and two 'induced' lines, and require that the patterns from the pairs of uninduced or induced lines agree or b) display PCR products from uninduced and induced lines over a time course of induction (5) On the relatively infrequent occasions when the initial display reveals a putative differentially-expressed mRNA, we repeat the PCR reactions with the appropriate 5' amplimer together with each of the individual 3' amplimers that were combined for ...

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