Faculty Bibliography

To assess the effect of the polyomavirus (Py) early proteins, the large T (LT), middle T (MT), and small T (ST) antigens, on gene expression from the Py late promoter, replication-defective plasmid constructs with the bacterial chloramphenicol acetyltransferase (cat) gene linked to this promoter were cotransfected into mouse or rat cells with plasmids capable of producing either LT, MT, or all three early proteins. When target CAT plasmids contained a truncated early region and thus had the coding potential for MT and ST, base-line CAT activities were low, whereas cotransfection with an LT plasmid resulted in up to 70-fold stimulation of CAT activity that was also reflected in similar increases in the level of steady-state mRNA. Studies with target plasmids with deletions within the Py regulatory region indicated that at least the major LT-binding site C and a functional enhancer region were both required for maximal stimulation of CAT activity. However, although enhancer deletions totally suppressed the ability of target plasmids to be trans activated, a consistent two- to fourfold stimulation of CAT activity by LT was still observed with a plasmid in which all three major LT-binding sites were deleted. Of four mutant LTs incapable of binding Py DNA but retaining immortalization potential, only one showed a low but significant trans-activating ability. When the early coding region was completely eliminated from the target plasmid, base-line CAT activity was increased 10-fold. LT failed to stimulate CAT activity to the same levels observed with target plasmid containing the truncated early region, but this limited response could be enhanced by supplying, in addition, MT and ST. Our results suggest that LT trans activation may involve the formation of a complex of transcriptional factors which interacts with the enhancer, an interaction that is facilitated both by the binding of LT to the Py regulatory region and by the presence of MT or ST or both, and that a significant portion of LT stimulation of late gene expression is a result of the removal of the competing early transcriptional unit via autoregulation. In addition, our results suggest that LT trans activation involves a second indirect component acting independently of LT binding and that the immortalization and trans activation functions of LT can be dissociated

We examined the binding of polyomavirus large (L-T)-, middle (M-T)-, and small-tumor antigens to DNA cellulose. At pH 6.0, the majority of L-T bound to calf thymus DNA cellulose, while little or no small tumor antigen was retained under these conditions. Unexpectedly, a small but reproducible proportion of M-T bound to both native and denatured DNA cellulose. M-T encoded by polyomavirus mutant dl 8, which expressed shortened L-T and M-T, bound to DNA, indicating that the deleted sequences are not required for DNA binding. Also, M-T from transformed BMT-1 rat cells, which synthesize exclusively this polyomavirus tumor antigen, bound to DNA, indicating that its binding is not due to association with other polyomavirus-encoded proteins. Using the DNA fragment immunoassay, we found that, under conditions in which L-T bound specifically to DNA fragments containing viral regulatory sequences, no viral DNA fragments were bound by M-T. The existence of distinct subpopulations of M-T that differ in their DNA-binding properties was indicated by rebinding experiments in which M-T that had bound to DNA cellulose rebound very efficiently, while that which had not been originally retained by DNA cellulose rebound poorly. Furthermore, the M-T-pp60 c-src complex did not bind to DNA cellulose. These data suggest that polyomavirus M-T is heterogeneous, consisting of populations of molecules that differ in their interactions with DNA cellulose

The c-myc oncogene has been implicated in the genesis of tumors as it has been found to undergo rearrangements and structural alterations in several types of neoplasms. However, the molecular mechanism of its involvement in transformation is still unclear as is the function of its protein product. We have studied the biological activity of the normal human c-myc oncogene in a well-characterized rat fibroblast line, F2408. The human gene, deleted of its 5' non-coding exon, was placed under the control of the Polyoma virus DNA regulatory region, linked to a dominant selectable marker (neomycin-resistance gene) and transfected into F2408 cells. Several neoR colonies were assayed for ability to grow in agar suspension, and the steady-state levels of mRNA transcripts of the exogenous c-myc gene were analyzed by Northern blot. While the greatest majority of these cell populations are phenotypically normal and express low levels of human myc transcripts, the rare neoR clones which display anchorage-independence express the human gene at high levels. This correlation was confirmed by the analysis of two neoR lines which contain low levels of c-myc RNA but gave rise to agar colonies at a frequency of about 10(-5). These agar derivatives were found to have greatly increased levels of c-myc mRNA when compared to the respective parental lines. The clones expressing high levels of myc RNA were found to be tumorigenic upon inoculation into young syngeneic rats. These results suggest that constitutive high expression of the normal c-myc gene is sufficient to confer anchorage-independence and tumorigenic potential to established rat fibroblasts

We have taken advantage of the inherent instability of integrated polyoma (Py) DNA sequences in the presence of a functional viral large T antigen (LT) to develop a eukaryotic host-vector system where copy number is controlled by temperature. A mouse cell line WOP32-4, that constitutively expresses a temperature sensitive (ts) LT, was transfected with plasmids containing the Py origin of DNA replication (ori) and either a neomycin-resistance gene (neo) or chloramphenicol acetyl transferase gene (cat) linked to the Py late promoter. Stable transformants were selected at 39 degrees C, the non-permissive temperature for the ts LT function. Upon shift to 33 degrees C, the resident Py sequences present in the WOP32-4 cells cannot excise due to an ori deletion. However, excision of the transfected plasmid molecules and subsequent extrachromosomal replication occur at high rates leading in some cases to the production of 1000-2000 copies per cell (average) of the plasmid. Proportional increases in either neo-specific mRNA or CAT activity were also observed. In situ hybridization for one cell line indicated that about 20% of temperature-shifted cells contained amplified plasmid DNA

In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neo-r colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 E1A core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression

We constructed and analyzed a series of deletion mutants in the noncoding regulatory region of tsa polyomavirus DNA to identify some of the sequences critical to the DNA replication origin and to the expression of the viral early genes in vivo. By using both transient and long-term assays under conditions where the influence of large T antigen (T-Ag) in replication or autoregulation was minimized, we observed no more than a 30% reduction in early gene expression upon removal of the CAAT or TATA elements or both. These assays demonstrated a predominant effect of upstream promoter or enhancer elements and indicated that removal of the CAAT or TATA boxes did not significantly affect viral early gene expression. Studies on the replicative ability of these mutants in mouse cells constitutively expressing the polyoma early proteins revealed that the removal of DNA sequences contained within a previously identified T-Ag high-affinity binding site (nucleotides 39 to 64) abolished viral DNA replication, whereas removal of two other high-affinity sites, closer to the early mRNA cap sites, did not. Furthermore, a deletion including this same high-affinity site plus a low-affinity binding site within the 32-base-pair palindrome of the origin core sequences eliminated the ability of the viral large T-Ag to efficiently repress early gene transcription. It is thus possible that the origin-proximal high-affinity T-Ag binding site is involved in both of the functions of large T-Ag, i.e., the initiation of viral DNA replication and the autoregulation of early gene transcription

We have examined the expression of chimeric plasmids containing coding sequences for the herpes simplex virus thymidine kinase (tk) gene or the Tn5 gene for neomycin resistance (neo) linked to the late promoter of polyoma DNA. Although polyoma late genes are generally not expressed in transformed cells containing only integrated viral DNA molecules, rat tk- or wild-type cells transfected with the tk- or neo-containing plasmids were capable of growing in medium containing either hypoxanthine-aminopterin-thymidine or G418, respectively, under conditions nonpermissive for extrachromosomal DNA replication, indicating that the tk or neo genes were fully expressed. Moreover, cells were capable of growth in either hypoxanthine-aminopterin-thymidine or G418, even in the absence of direct selection for this activity. Northern analysis indicated steady-state levels of tk or neo transcripts that approximated the levels of polyoma early transcripts. S1 analysis showed that these transcripts initiated within the late promoter of polyoma and that their 5' ends mapped at positions similar or identical to those utilized during late lytic infection. The effect of substitution of polyadenylation signals was examined. Although plasmids containing the polyoma early polyadenylation signal were more efficient in conferring to cells a stable G418-resistant phenotype than similar constructions using the late signal, both signals were found to be effectively utilized. This indicates that the inability to detect late transcripts in polyoma-transformed cells in the absence of free viral DNA production is not an effect of inefficient mRNA cleavage or polyadenylation. Our results suggest that late gene expression in integrated polyoma genomes is not regulated at the level of message initiation but, most likely, through posttranscriptional events

We describe here a new cell line, EL2, which spontaneously arose from primary rat embryo fibroblasts and has the distinctive property of being highly susceptible to a number of different transforming genes. The high susceptibility is expressed not only in high transformation frequencies but, most importantly, in an unusually high rate of growth of EL2 transformants under selective conditions, i.e., in soft agar or as foci. The biological characteristics of EL2 cells greatly accelerate the isolation of transformants from known oncogenes and could be useful to detect new transforming genes

The evolution of mouse cells transformed by a recombinant plasmid containing the genome of the tsA mutant of polyoma virus (Py) cloned at the BamHI site into the plasmid pML, whose sequences therefore interrupt the Py late region, has been studied. Clones of transformed cells were selected at 39 degrees (nonpermissive temperature for large T antigen). Under these conditions viral DNA integration is stable and the cells display a uniformed transformed phenotype. Also studied in detail was the evolution of one of these cell lines (A4) upon shift to a temperature permissive for large T-Ag function (33 degrees); immediately after shift, 90% of the population became intensely positive for T-Ag and a considerable amount of free-viral DNA was produced, accompanied by a clear cytopathic effect. Surviving cells proliferated actively after 4 weeks at 33 degrees and showed a decreased expression of large T-Ag (only 2-3% of the population was T-Ag positive by immunofluorescence), a drastic reduction in the amount of free-viral DNA produced, but no apparent change in the pattern of integration of Py DNA in the host chromosomes. Analysis of the high-molecular-weight DNA with the restriction enzymes HpaII and MspI revealed that the cytosines in the recognition sequences of these enzymes were methylated. Accordingly, treating the cells with 5-Azacytidine, a methylation inhibitor, results in the expression of viral T-Ags in more than 80% of the cell population. Analysis of DNA transcription revealed a dramatic reduction of virus-specific poly(A)+ mRNA in the methylated cells; in addition, the phenotype of the 33 degrees A4 populations was much less transformed than that of the original cultures. The block of Py expression by methylation is not complete; approximately 2% of the cells remain T-Ag positive and viral transcription is not completely suppressed. This could be explained by an incomplete methylation which randomly leaves unmethylated sequences essential for Py gene expression, or by the fact that methylation is not sufficient to block transcription completely. Possible mechanisms underlying this type of evolution are discussed