Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

Chromosome analyses and anchorage-independent growth of SV40-induced morphologically transformed epithelial cells from amniotic fluids

Summary Epithelial cells from amniotic fluid cell cultures are morphologically transformed by simian virus 40, 20 to 30 d after infection. The cells of the transformed colonies are highly basophilic, have a high nuclear-to-cytoplasmic ratio, and show a dense growth pattern. The cells are virus producers, and ultimately, after continuous passage, the cell lines reach a crisis situation with no growth. Twelve morphologically transformed cell colonies were isolated from five different individuals for chromosome analyses after approximately 18 population doublings (second bottle passage). For all cell lines diploid cells were observed. Banding of the chromosomes revealed normal morphology of euchromatic and heterochromatic regions. The suggestion is made that chromosome alteration is not necessary, nor a prerequisite, for the morphologically transformed phenotype to be expressed and that the transformation process per se causes chromosomal instability. Tests for colony formation of the 12 cell lines in semisolid medium showed that different transformed colony isolates from the same individual donor of the cells either formed or did not form colonies in agar. The size of the colonies was also consistent within individuals as compared to between individuals. These limited results are suggestive of a dependence upon the genetic constitution of the individual donor of the cells for colony formation in soft agar. Supported by National Science Foundation Grant PCM77-15876.     

Association of SV40 DNA sequences with nuclear matrix in SV40 transformed hamster fibroblasts

Nuclear matrices were isolated by the high-salt, non-ionic detergent method from SV40-transformed hamster fibroblasts (TSV5 cell line), and from hamster tumours derived from these cells. DNA isolated from matrices and total nuclei was hybridized with nick-translated SV40 DNA. The enrichment of matrix DNA with SV40 DNA sequences was observed in all five experiments with matrix DNA of TSV5 cells but only in five out of nine matrix DNA isolated from tumour cells.     

Nucleotide sequence-directed mapping of the nucleosomes of SV40 chromatin

In our previous work we have shown by comparison of experimental and computational data that the positions of the histone octamers bound to the DNA molecule appear to be completely sequence-dependent. This provides a convenient and quick method for locating the nucleosomes along the DNA molecule, as soon as the nucleotide sequence is known. Using this computational approach, the complete nucleosomal map of the SV40 minichromosome has been constructed. The map consists of 25 nucleosomes, with their coordinates (centers) being specified with high accuracy. The map is found to be in remarkable agreement with available experimental data.     

Endogenous butyrylcholinesterase in SV40 transformed cell lines: COS-1, COS-7, MRC-5 SV40, and WI-38 VA13

Summary Comparison of proteins expressed by SV40 transformed cell lines and untransformed cell lines is of interest because SV40 transformed cells are immortal, whereas untransformed cells senesce after about 50 doublings. In MRC-5 SV40 cells, only seven proteins have previously been reported to shift from undetectable to detectable after transformation by SV40 virus. We report that butyrylcholinesterase is an 8th protein in this category. Butyrylcholinesterase activity in transformed MRC-5 SV40 cells increased at least 150-fold over its undetectable level in MRC-5 parental cells. Other SV40 transformed cell lines, including COS-1, COS-7, and WI-38 VA13, also expressed endogenous butyrylcholinesterase, whereas the parental, untransformed cell lines, CV-1 and WI-38, had no detectable butyrylcholinesterase activity or mRNA. Infection of CV-1 cells by SV40 virus did not result in expression of butyrylcholinesterase, showing that the butyrylcholinesterase promoter was not activated by the large T antigen of SV40. We conclude that butyrylcholinesterase expression resulted from events related to cell immortalization and did not result from activation by the large T antigen.     

Studies of simian virus 40 DNA. VII. A cleavage map of the SV40 genome

A physical map of the Simian virus 40 genome has been constructed on the basis of specific cleavage of Simian virus 40 DNA by bacterial restriction endonucleases. The 11 fragments produced by enzyme from Hemophilus influenzae have been ordered by analysis of partial digest products and by analysis of an overlapping set of fragments produced by enzyme from Hemophilus parainfluenzae. In addition, the single site in SV40 DNA cleaved by the Escherichia coli R_I restriction endonuclease has been located. With this site as a reference point, the H. influenzae cleavage sites and the H. parainfluenzae cleavage sites have been localized on the map.     

Analysis of stable and unstable viral forms in SV40-infected human keratinocytes

We analyzed the state of the genomic DNA of the papovavirus SV40 in human keratinocytes as viral-infected cells gradually acquired a transformed phenotype over time. Initially, the vast majority of the viral DNA is maintained either in a full-length supercoiled form or as truncated subgenomic fragments with little evidence of integration. However, analyses of clonal populations revealed great heterogeneity and instability of the viral DNA, and we were able to isolate one clonal subpopulation in which integrated forms of the virus appeared to predominate. Similarly, uncloned populations eventually ceased production of the "free" viral DNA after several years in culture and instead came to display tandemly repeated SV40 copies at a single host integration site. Interestingly, Bg1 II digestion of host DNA generated restriction fragments containing the integrated SV40 DNA, which were of differing sizes in cultures at the 144th vs the 163rd serial passage suggesting modification or rearrangement of sequences at or near the integration site. Host sequences flanking the integrated viral DNA at the 163rd serial passage have been isolated on restriction fragments generated by Eco RI, Bam HI, and Hpa II digestion. These analyses suggest that the integrated virus is linearized near the Bg1 I site and contains a large deletion in the SV40 early region at one of the viral-host junctions.     

The induction of growth arrest in fibroblasts by SV40 T antigen

DNA tumor viruses such as SV40, Ras and papillomaviruses are the most commonly used agents in immortalization of non-hematopoietic cells, but the results are quite different. Some of them even lead instead to a senescence-like state. To verify the potential of SV40 T antigen-mediated immortalization or properties and functions of it to regulate cell growth, human dermal fibroblasts were cultured and then transfected with eukaryotic expressing plasmid psv3-neo which containing SV40 T DNA. We found that expression of oncogenic SV40 T in human dermal fibroblasts resulted in growth, arrest, earlier than the occurrence of control cell senescence, although telomerase was positive and cells grew faster than control ones in early stage following transfection. These observations suggest that SV40 T antigen can activate growth arrest in human dermal fibroblasts under normal growth condition instead of always prolonging the lifespan of fibroblasts. Moreover, high rate of cell division in early stage after transfection may be associated with the expression of telomerase activity.     

Comparison of spontaneous and ultraviolet-induced mutagenesis on naked SV40 DNA and SV40 virus

Molecular aspects of mutagenesis in mammalian cells have been essentially analyzed using biological probes such as viruses and shuttle vector. Although the main data concerning the specificity of carcinogen-induced mutations are similar, the observed spontaneous mutation frequencies are significantly different when using one or the other model. This frequency is considerably higher with shuttle vectors than with viruses. We have performed an analysis of mutagenesis in order to determine if the obligatory transfection step associated with shuttle vector technology was responsible for the high mutation frequency found with these molecules. For this purpose simian virus 40 (SV40) genome used as virus or as naked DNA was introduced into permissive cells by viral infection or DNA transfection respectively. Our results show that transfection alone does not induce a higher mutation frequency on SV40 DNA the virus infection. Moreover, we have shown that the ultraviolet-light induced mutation spectrum was similar on the SV40 VP1 gene after viral infection or DNA transfection.     

Complete Genomic Sequence Analysis of a New SV40 Isolate

The genome of a new SV40 strain (SV-IMB) isolated from a rhesus monkey was completely sequenced and compared with other isolates. The results showed that the whole genome contains 5246bp, and the average identity of SV-IMB was 98.1% as compared to other SV40 isolates. Its regulatory region is composed of a complete enhancer and a defective enhancer. Amino acid changes occurred to some extent in both the large T antigen (T-Ag) and VP1 region. The findings demonstrate that the SV-IMB is a new SV40 isolate.     

Persistence of phage lambda DNA in genomes of mouse cells transformed by lambda-carrying SV40 vectors.

To test the suitability of simian virus 40 (SV40) DNA as a vector for inserting DNA segments into the chromosomes of mammalian cells, an EcoRI-A fragment of bacteriophage lambda DNA was covalently joined to a fragment of SV40 DNA and used to transform mouse cells in culture. Three independent, morphologically transformed clones were obtained that were positive for SV40 T-antigen by immunofluorescence staining. DNA from each transformant was examined by restriction enzyme analysis and found to contain both lambda and SV40 sequences. Co-migration of some fragments containing lambda and SV40 sequences following digestion of transformed cell DNA by each of four different restriction enzymes indicated that part of the retained lambda and SV40 DNA was linked in two of the three lines. In the third line, however, none of the restriction fragments had both lambda and SV40 sequences. Although the presence of non-integrated lambda DNA was not excluded, at least some of the lambda DNA appeared to be linked to host cell DNA. Results of digestion by EcoRI suggested that in some cases the transforming linear molecule had probably circularized prior to integration.     

Detection of SV40 DNA sequences in malignant mesothelioma specimens from the United States, but not from Turkey

The incidence of malignant mesothelioma (MM) shows a strong epidemiological association with exposure to asbestos fibers. Recently, simian virus 40 (SV40) DNA sequences have been reported in MM tumor specimens from the United States and several European countries, and the SV40 tumor virus has been implicated as a potential co-factor in the etiology of this disease. However, several large studies from the US, Finland, and Turkey did not detect SV40 sequences in MM samples. To address this discrepancy, MM specimens from Turkey and the US were analyzed in the same laboratory under identical conditions to detect the presence of SV40 DNA. We detected SV40 sequences in 4 of 11 specimens from the United States, but in none of the 9 Turkish samples examined. These findings suggest that geographical differences exist with regard to the involvement of SV40 in human tumors.     

Complete genomic sequence analysis of a new SV40 isolate

The genome of a new SV40 strain(SV-IMB) isolated from a rhesus monkey was completely sequenced and compared with other isolates. The results showed that the whole genome contains 5246bp, and the aver age identity of SV-IMB was 98.1% as compared to other SV40 isolates. Its regulatory region is composed of a complete enhancer and a defective e enhancer. Amino acid changes occurred to some extent in both the large T antigen (T-Ag) and VP1 region. The findings demonstrate that the SV-IMB is a new SV40 isolate.     

Tumorigenicity of revertants from an SV40-transformed line

A syndrome of in vitro properties correlates with the tumorigenicity of SV40-transformed rodent cells. These properties are plasminogen activator production, loss of large actin cables, and anchorage-independent growth. An established rat fibroblast line, its SV40 transformant, several T-antigen negative revertants, and a spontaneous retransformant isolated form one of the revertants were analyzed in vivo for their tumorigenicity and in vitro for the syndrome. The two transformed lines were highly tumorigenic, and had clearly abnormal in vitro properties. The parental rat line was weakly tumorigenic in nude mice and demonstrated a slightly transformed response in the in vitro assays. The revertants were completely nontumorigenic. Expression of the in vitro syndrome was not uniform for all revertants; however, most cell lines maintained the correlation of the syndrome and tumorigenicity.     

Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells

A variety of methylated oligonucleotides were derived from mouse L cell messenger RNA and heterogeneous nuclear RNA by digestion with specific ribonucleases, and the cap-containing oligonucleotides separated from those containing internal m⁶A by chromatography on diborylaminoethyl-cellulose. Cap-containing sequences of the type m⁷GpppX^mpG, m⁷GpppX^mpY^(m)pG, m⁷GpppX^mpY^(m) pNpG and m⁷GpppX^mpY^(m)p(Np)_{> 1}G have distinctive non-random compositions of the 2′-O-methylated constituent X^m; yet sequences of a particular type and composition occur with a remarkably similar frequency in mRNA and hnRNA². For example, approximately 20% of the cap sequences in both hnRNA and mRNA are m⁷Gppp(m⁶)A^mG, whereas less than 1% are m⁷GpppU^mpG. The high degree of similarity in cap sequences is consistent with the previously postulated precursor-product relationship between hnRNA caps and mRNA caps.The composition of the Y position in capped hnRNA molecules was determined to be (29% G, 20% A, 51% Py), which differs considerably from the composition of Y^m in the cap II forms of mRNA (8% G^m, 11% A^m, 81% Py). Given the precursor-product relationship between hnRNA caps and mRNA caps, this result provides strong evidence that only a restricted subclass of mRNA molecules receive the secondary methylation at position Y.In both hnRNA and mRNA the internal m⁶A occurs in well-defined sequences of the type: $\text{-N}{}_1\text{-(}\text{G}\text{A}\text{)-m}{}^6\text{A-C-N}{}_2\text{-}$, the 5′ nearest-neighbor of m⁶A being G in about three-quarters of the molecules and A in about one-quarter of the molecules. The nucleotide N¹ is a purine about 90% of the time and the nucleotide N² is rarely a G. These same sequences are present in large (> 50 S), as well as small (14 S to 50 S) hnRNA. These results raise the possibility that the internal m⁶A, like caps, may be conserved during the processing of large hnRNA into mRNA. Two models based on this idea are discussed.