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A Cowie

Publications and source records attributed to A Cowie.

At least 37 records · Page 2Linked to original sources
PubMed1986

Regulation of polyomavirus late promoter activity by viral early proteins.

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.

Antigens, Polyomavirus Transforming↗
PubMed1986

Immortalization of rat embryo fibroblasts by mutant polyomavirus large T antigens deficient in DNA binding.

We have identified a putative DNA-binding domain in polyomavirus large T antigen. Mutations introduced into the gene between amino acids 290 and 310 resulted in proteins that no longer bound to the high-affinity binding sites on the polyomavirus genome, showed no detectable nonspecific DNA binding, and were not able to initiate DNA replication from the viral origin. These mutant T antigen genes were introduced into rat embryo fibroblasts together with the neomycin resistance gene to allow selection for growth in the presence of G418. All the mutations tested facilitated the establishment of these cells in long-term culture at an efficiency indistinguishable from that of the wild-type protein.

Amino Acid Sequence↗
PubMed1986

Binding of polyomavirus large T antigen to the human hsp70 promoter is not required for trans activation.

Polyomavirus large T antigen binds to two sites located between positions -110 and -170 of a human heat shock protein 70 (hsp70) promoter. Methylation interference studies show that binding for each site is determined by two GPuGGC pentanucleotide sequences. The specificity of this binding interaction is similar to that observed for large T binding to the viral genome. The existence of sequences that bind a viral protein in a cellular promoter raises the possibility that these sequences play a role in gene expression in an uninfected cell. We show that hsp70 large T antigen binding site 1 is capable of functioning as an upstream promoter element in cells that do not contain any viral T antigen. Genetic analysis of this effect suggests that a cellular factor exists that has a binding specificity that overlaps but is not identical to that of polyomavirus large T antigen. To determine whether binding of polyomavirus large T antigen can regulate expression of the intact human hsp70 promoter, we have introduced the promoter into mouse cells with plasmids that express the polyomavirus early proteins. These proteins stimulate the level of correctly initiated hsp70 transcripts, but surprisingly the degree of stimulation remains unchanged for promoter constructs in which the large T antigen binding sites have been deleted. These observations suggest that trans activation of the hsp70 promoter by the polyomavirus early proteins occurs through protein-protein interactions and not through sequence-specific DNA binding.

Acetyltransferases↗
PubMed1984

DNA binding activity of polyoma virus large tumor antigen.

Polyoma virus large tumor antigen from productively infected mouse cells has been purified to greater than 50% homogeneity by a simple immunoaffinity procedure using monoclonal antibodies. A radioimmunoreaction was devised for assaying purity. The purified large tumor antigen retained its antigenicity and its ability to bind DNA specifically. The regions on the polyoma virus genome recognized by the protein were characterized. Three binding regions were localized within the portion of the genome between the viral origin of DNA replication and the protein coding sequence, overlapping the early promoter and the sites of initiation of mRNAs that specify the viral tumor antigens. The binding regions each contain direct repeats of the pentanucleotide sequence G-R-G-G-C.

Animals↗
PubMed1984

Construction and functional characterization of polyomavirus genomes that separately encode the three early proteins.

Modified polyomavirus genomes that individually encode the large and small T proteins were constructed by exchanging restriction endonuclease fragments between cDNA copies of the respective mRNAs and cloned genomic DNA. The efficacies of the new constructs, and that of the middle T protein gene described previously (R. Treisman , U. Novak, J. Favaloro , and R. Kamen , Nature [London] 292:595-600, 1981), were demonstrated with simian virus 40 (SV40)-polyomavirus recombinants in which part or all of the SV40 late region was replaced with the modified polyomavirus early genes. Each of the three recombinant viruses induced the synthesis of only the expected polyomavirus early protein in infected CV-1 cells. The rates of synthesis of large, middle, and small T proteins were ca. 1.5, 4.0, and 9.0 times the rate of synthesis of SV40 large T protein, respectively. The deletion of introns had no detrimental effect on mRNA biogenesis. Indeed, a further polyomavirus-SV40 recombinant, containing wild-type polyomavirus early region DNA, expressed an aberrant 58,000-dalton form of the middle T protein which we believe to result from utilization of a cryptic splice site. Immunofluorescence studied with monkey cells infected by the recombinant viruses allowed us to determine the cellular locations of the polyomavirus early proteins. Overproduction of the middle T protein did not result in a corresponding overproduction of the middle T protein-associated tyrosine phosphokinase activity.

Animals↗
PubMed1984

Multiple binding sites for polyomavirus large T antigen within regulatory sequences of polyomavirus DNA.

Polyomavirus large T antigen binds specifically to multiple sites within the regulatory region of the viral genome. Experiments done with crude extracts from wild-type virus-infected mouse cells and immunoprecipitation of protein-DNA complexes localized two high-affinity binding sites on the early region side of the DNA replication origin. Purification of the large T antigen by immunoaffinity chromatography made it possible to refine the analysis through application of DNase I footprinting. The high-affinity interactions were resolved into three closely spaced, but distinct, binding regions. These begin at a site only slightly overlapping the early boundary of the core replication origin, a location highly homologous to that of simian virus 40 large T antigen-binding site I, but then extend away from the origin toward the early coding sequence and thus span the early region transcriptional initiation sites. Each tight-binding region contains from two to four copies of the sequence 5'-(A = T)G(A greater than G)GGC-3' repeated at 9- to 11-base-pair spacing. At high protein concentrations and at low ionic strength, additional sites within the core replication origin and in the enhancer region were protected from DNase I digestion. These minor binding sites also included repeats of sequences related to the consensus, but at different spacings. Our results suggest that, unlike simian virus 40 DNA, the polyomavirus genome may have distinct regions of interaction with its large T antigen which separately are involved in initiation of DNA replication and the regulation of viral transcription.

Antigens, Viral, Tumor↗
PubMed1983

Expression of the large T protein of polyoma virus promotes the establishment in culture of "normal" rodent fibroblast cell lines.

Transfer into mouse and rat embryo fibroblasts in primary culture of cloned polyoma virus genes encoding only the large T protein led to the establishment of flat colonies in sparse subcultures at a frequency equal to that of transformation by wild-type virus. Cell lines could be derived from such colonies and maintained in culture for large numbers of generations without entering crisis. They exhibited a normal phenotype, by the criteria of growth on plastic to a low saturation density and of anchorage dependency. However, they required a lower serum concentration for growth than spontaneously established 3T3 cells. Similar results were obtained after transfer of recombinant DNA molecules encoding only the amino-terminal 40% of the large T protein, suggesting that this "immortalization" function corresponds to the activity of an amino-terminal domain of the protein. Immunoprecipitation analysis of T antigens in cell lines established after transfer of the full-size and of the truncated large T genes demonstrated the expression of the full-size large T protein and of a Mr 40,000 antigen expressed from the amino-terminal part of the gene, respectively. After transfer of a "large T only" plasmid that carries a tsa mutation, cell lines were established at 33 degrees C with the same efficiency as with the wild-type large T gene, but their growth was arrested after a shift to 40 degrees C, with a progressive loss in cell viability. This result indicates a continuous requirement for a large T function in the maintenance of "immortality."

Animals↗
PubMed1982

The roles of individual polyoma virus early proteins in oncogenic transformation.

The expression in normal rat cells of modified polyoma virus genomes, separately encoding large T, middle T or small T antigens, has allowed the investigation of the roles of these proteins in oncogenic transformation. Middle T is sufficient to transform cells of established lines but the transformants are serum dependent. Large T lacks intrinsic oncogenic potential but can relieve the serum dependence of normal and transformed cells. Middle T alone cannot transform primary rat embryo fibroblasts.

Animals↗
PubMed1982

Comparison of the polyoma virus early and late promoters by transcription in vitro.

Polyoma virus DNA was transcribed in the HeLa whole cell extract in vitro system (1). Early region transcripts with the same 5'-ends as in vivo mRNAs, located 31 +/- 2bp from 'TATA'-boxes, were synthesized by RNA polymerase II. Sequences sufficient for efficient expression of the early promoter were present in a substitution mutant lacking viral DNA from a position 55bp before the principal cap sites. Late region transcripts were synthesised inefficiently. Only one (at nt5129 +/- 2) of the many late mRNA cap sites functioned as an in vitro initiation point. This was the one 5'-end located 31 +/- 2bp from a sequence resembling the 'TATA' consensus. The proportion of late to early region RNA polymerase II transcripts decreased dramatically at suboptimal template concentrations. An hypothesis to explain the regulation of late gene expression in vivo based on these results is proposed. A linear templates were transcribed only by RNA polymerase II, transcripts with the same sense as late mRNAs and 5'-ends at nt5076 +/- 2 were produced from superhelical template by an alpha amanitin resistant enzyme.

Base Sequence↗
PubMed1982

DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter.

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.

DNA, Viral↗
PubMed1981

Sequences at the capped 5'-ends of polyoma virus late region mRNAs: an example of extreme terminal heterogeneity.

We have localized with respect to the genomic DNA sequence the capped 5'-termini of polyoma virus late region mRNAs. A minimum of fifteen different purine termini were found within a 94 base pair region (66.36 to 68.12 map units, nt 5075-5168) immediately preceding the sequence determining the late region mRNA leader repeat (1-3). The most common termini occur at nearly every possible purine within a 25 bp sequence proximal to the leader repeat unit. These do not bear the usual positional relationship to a sequence resembling the 'TATA' box consensus. Deletion mutants lacking minor cap sites and sequences upstream from the principal cap sites were viable. A deletion mutant lacking one of the principal cap sites formed small plaques, while a slightly larger deletion further impinging on the principal cap site region was non-viable. The principal cap sites, which we assume to be transcriptional initiation points, are included in a DNaseI hypersensitive region of polyoma virus chromatin (4).

Animals↗
PubMed1981

The structures of the spliced mRNAs encoding polyoma virus early region proteins.

The polyoma virus early region mRNAs synthesized during productive infection of mouse cells have been characterized at the nucleotide level. One- and two-dimensional agarose gel fractionation of nuclease S1-resistant RNA-DNA hybrids was used to establish basic structures. The two splice donors and the two splice acceptors were positioned more precisely by high resolution S1-gel mapping with terminally labeled DNA probes and polyacrylamide gels. The nucleotide sequences across the three splice joints were established by cloning and sequencing partial cDNA copies of the mRNAs. In combination with data on the polyadenylated 3'-end previously published, and the detailed analysis of the capped 5'-ends presented elsewhere, the present data complete the description of a family of differentially spliced mRNAs able to encode the known early region gene products, small-T, middle-T, and large-T proteins.

Animals↗
PubMed1980

Localization of three major cappe 5' ends of polyoma virus late mRNA's within a single tetranucleotide sequence in the viral genome.

The nucleotide sequences at the 5' ends of polyoma virus late mRNA's were determined by direct RNA sequencing of decapped and 5'-(32)P-labeled RNase T1 oligoribonucleotides. Virus-specific polyadenylated cytoplasmic RNA, which was isolated from mouse cells late during productive infection, was enzymatically or chemically treated to specifically remove the cap structure (m(7)Gppp). The unblocked 5' ends of the viral mRNA's were then labeled enzymatically with (32)P, and the RNAs were digested with RNase T1 and fingerprinted. Three oligonucleotides derived from capped termini were isolated, and their sequences were determined to be pGmACAUUUUCUAUUUUAAGp, p(m)AmCAUUUUCUAUUUUAAGp, and p(m)AmUUUUCUAUUUUAAGp. These oligonucleotides comprise a staggered set with members 15, 17, and 18 nucleotides long, which share a common 3' sequence for 15 nucleotides. The sequences correspond exactly to the polyoma virus DNA sequence (Arrand et al., J. Virol. 33:606-618, 1980) from 66.79 to 66.46 map units (between 75 and 92 nucleotides preceding the ATG initiation codon for the capsid protein VP2). Previous results showed that the sequence between 13 and 64 nucleotides preceding the VP2 initiation codon corresponds to oligonucleotides reiterated in the leader sequence which is spliced onto the bodies of the three functionally distinct viral late mRNA's (Flavell et al., Cell 16:357-372, 1979; Legon et al., Cell 16:373-388, 1979). The three capped oligonucleotides we sequenced are derived from the first large predicted T1 oligonucleotide 5' to those detected in the leader sequence. The occurrence of a cap at each purine of a single tetranucleotide sequence reflects micro-heterogeneity either in transcriptional initiation or in processing cleavage involved in cap syntheses.

Base Sequence↗
PubMed1979

Multiple 5' terminal cap structures in late polyoma virus RNA.

Nuclear and cytoplasmic polyoma virus-specific RNA extracted from 32P-labeled mouse embryo cells late during productive viral infection was analyzed for the presence of 5' terminal capped structures by complete digestion with RNAases T1, T2 and A, followed by two-dimensional electrophoretic fractionation. Seven major cap I structures (m7 GpppNm1pN2p) were observed in both cases. These termini were further characterized by digestion with penicillium nuclease P1, followed by product analysis in a variety of alternative separate systems. Each structure had an individual combination of N1 and N2 nucleotides, where N1 was always a purine nucleotide but N2 was any nucleotide subject to the single exception that m7GpppGmpCp is found only in low yield. Four different cap II derivatives (m7GpppNm1pNm2pN3p) of four of the cap I structures were also detected in cytoplasmic RNA. None of the termini described derived from contaminating host cell RNA. All of these cap structures mapped on the polyoma viral DNA genome between 66 and 71 map units, a region distant from the 5' end of the bodies of two of the three late polyoma mRNAs. All the polyoma virus-specific cap structures, however, were present in each of the purified 16S, 18S and 19s late mRNAs. These data suggested that families of capped leader sequences of varying sizes are attached to the main body of each late polyoma mRNA species by a splicing mechanism.

Animals↗
PubMed1979

Amplification in the leader sequence of late polyoma virus mRNAs.

Ribonuclease T1 fingerprints of the three "late" polyoma virus mRNAs show that oligonucleotides of the leader sequence are present in multiple copies in each mRNA. These oligonucleotides, however, appear unimolar in fingerprints of complete, continuous transcripts of the late strand of the viral DNA. Oligonucleotides which are represented only once in the DNA are thus reiterated in the mature mRNAs. Consequently, when mRNA was hybridized to the leader region of immobilized viral DNA, those copies present in excess of their genomic representation failed to hybridize and were released by RNAase treatment. Analysis of the RNAase-resistant hybrids revealed a series of leader species with complex sequence arrangements. We suggest that these complicated reiterated sequences are generated during the processing of a precursor RNA which extends several times around the genome. This RNA would be shortened by a series of splicing reactions which conserve sequences from the leader region and attach them to a suitable coding sequence.

Animals↗