SV40 DNA Fragments Research Articles

Efficient gene transfer to hematopoietic progenitor cells using SV40-derived vectors.

We used recombinant SV40 (rSV40)-derived vectors to deliver transgenes to human and simian hematopoietic progenitor cells in culture, and in vivo after transduction ex vivo. rSV40 are highly efficient vectors that are made in very high titers. They infect almost all cells, whether resting or dividing. Two rSV40s were used: SV(HBS), carrying hepatitis B surface antigen as a marker; and SV(Aw) carrying IN#33, a single chain Fv antibody against HIV-1 integrase. CD34+ cells derived from human fetal bone marrow (HFBM) and rhesus macaque bone marrow were transduced once with SV(HBS) without selection. On average 60% of colonies derived from transduced CD34+ cells carried and expressed HBsAg, as assessed by PCR and immunochemistry. Transgene carriage persisted following differentiation of transduced rhesus CD34+ cells into T lymphocytes. In an effort to increase the percentage of gene-marked cells, three sequential treatments of CD34+ cells were done using SV(Aw), without selection. Two weeks later, >95% of colonies expressed IN#33. Unselected SV(Aw)-transduced CD34+ cells from HFBM were transplanted into sublethally irradiated SCID mice. Bone marrow harvested 3 months later showed that >50% of bone marrow cells expressed IN#33. This is comparable with the percentage of human cells in these animals' bone marrow as judged by immunostaining for human CD45. The stability and longevity of transduction in this setting suggests that rSV40 vectors integrate into the cellular genome. This possibility was supported by finding that PCR of genomic DNA using primer pairs with one cellular and one viral primer yielded PCR products only in transduced, but not control, cells. These PCR products hybridized with an SV40 DNA fragment. Thus, rSV40 vectors transduce normal human and primate bone marrow progenitor cells effectively without selection, and maintain transgene expression in vivo following reimplantation. Such high efficiency transduction may be useful in treating diseases of CD34+ cells and their derivatives.

Experimental evidence for RNA trans-splicing in mammalian cells.

We present evidence that mammalian cells have the ability to generate functional mRNA molecules by trans-splicing. Rat cells, transformed by an early SV40 DNA fragment (Bst/Bam) synthesize a truncated T antigen (T1 antigen), although the cells do not have a direct sequence homology for the T1 antigen at the DNA level. The Bst/Bam DNA fragment encodes exclusively for the second SV40 T antigen exon (aa 83-708) and contains the entire small t antigen intron. To synthesize the corresponding mRNA (T1 mRNA), the cells utilize a cryptic 5' splice site within the second exon (codons for aa 131/132) as donor site and the upstream small t antigen 3' splice site as the acceptor site. Since these sites are in an inverted order on the pre-mRNA, two Bst/Bam transcripts are required to generate one T1 mRNA molecule. HeLa cell nuclear extracts also performed the trans-splicing reaction in vitro.

Detection of allergen- and mitogen-induced human cytokine transcripts using a competitive polymerase chain reaction

Human cytokines, IL-4, IL-5, and IFN-γ play an important role in the regulation of IgE synthesis and atopic diseases. In this communication, we describe the development of a quantitative assay of steady-state cytokine mRNAs (IL-4, IL-5, and IFN-γ) from a variety of cell sources, including peripheral blood mononuclear cells (PBMCs) stimulated with either a mitogen (PHA) or ragweed pollen allergen extract, and cells from allergen-challenged inflammatory sites. Quantitative analysis of IL-5, IL-4 and IFN-γ transcripts was achieved by a competitive reverse transcription-polymerase chain reaction (RT-PCR) technique using internal standard (IS)_cRNAs in the presence of specific oligonucleotide primers. Each IS was generated from a plasmid vector contining the respective cytokine cDNA modified by insertion with an SV40-DNA fragment. Both test RNA and IS were reverse-transcribed and subjected to the ‘competitive’ PCR in the same tube. We first demonstrate the linearity and reproducibility of this technique; second, we apply this competitive PCR assay to analyze quantitatively the expression of IL-4, IL-5, and IFN-γ transcripts in PBMCs before and after stimulation with PHA or crude ragweed allergen. Finaly, we analyzed cells isolated from the lung lavage fluids of an atopic subject following allergen challenge, and showed a significant increase of IL-4 and IL-5 transcripts, but not IFN-γ, in the allergen-challenged site when compared to the control. This technique of PCR quantitation provides an easy and efficient tool to study the expression of cytokine genes in allergic inflammatory diseases.

Effects of base mutations on topoisomerase II DNA cleavage stimulated by mAMSA in short DNA oligomers.

DNA cleavage by topoisomerase II in the absence or presence of mAMSA, and VM-26 was investigated in a series of oligonucleotides of 36 and 42 base pairs, which were derived from the DNA sequence of the major topoisomerase II cleavage site in the matrix-associated region of SV40 DNA. Topoisomerase II introduced strand cuts at several sites in the oligonucleotides, and the sequence selectivities of DNA cleavage with and without drugs were the same as in larger SV40 DNA fragments. A time course analysis showed that mAMSA specifically stimulated DNA cleavage at the 4263/4266 site, while DNA cleavage was specifically induced at the 4265/4268 site by the enzyme without drug or with VM-26. In agreement with recent findings on local nucleotide requirements in order for mAMSA to stimulate DNA cleavage, the 4263/4266 site had adenines at the two positions +1. This nucleotide requirement was challenged by mutating the bases 4263 and 4266 of the oligonucleotide representing the natural SV40 DNA sequence. New cleavage sites were not observed in the mutated oligonucleotides, and base mutations had an effect on DNA cleavage induced with and without the two drugs. This general effect was likely due to the sensitivity of topoisomerase II itself to the local DNA sequence. Nevertheless, effects of base mutations were more pronounced for mAMSA than for VM-26. Point mutations of either base 4263 or 4266, representing the two positions +1, reduced markedly the stimulative effect of DNA cleavage at the 4263/4266 site by mAMSA, and mutations of both bases completely abolished it.(ABSTRACT TRUNCATED AT 250 WORDS)

SV40 recombinants carrying a d(CT · GA) 22 sequence show increased genomic instability

Repetitive d(CT · GA) n sequences are commonly found in eukaryotic genomic DNA. They are frequently located in sites involved in genetic recombination or in promoter regions. To test for their possible biological function, a d(CT · GA) 22 synthetic sequence was introduced into the genome of SV40, since it constitutes an appropriate model system for eukaryotic chromatin. When SV40 infects permissive cells, it proliferates in the form of a minichromosome. The simple repetitive sequence indicated above was inserted at the unique HpaII site of SV40 (at nt 346), and the genomic stability of SV40 recombinants carrying the d(CT · GA) 22 sequence (SV/CTβ viruses) was analyzed. Upon serial passage through permissive CV1 cells, SV/CT 22 recombinants show an increased production of defective viruses. Generation of SV/CT 22 variants is likely to take place via recombination between and within viral molecules. The enhancement of the rate of recombination induced by the repetitive sequence is likely to be related to its known propensity to form triple-stranded structures. Many different variants coexist in the same viral population indicating that the mechanism by which they are produced is not unique. One variant (SV/X), showing a replicative advantage, was characterized in detail. Variant SV/X accounts for a large proportion of the total viral population. Its genomic organization corresponds to a tandem duplication of an early SV40 DNA fragment spanning from approx. nt 3200-nt 160. Variant SV/X contains a duplicated SV40 ori.

Phosphorylation of mammalian DNA topoisomerase I and activation by protein kinase C.

The influence of mammalian DNA topoisomerase I phosphorylation on enzyme activity has been investigated. Dephosphorylation by calf intestine alkaline phosphatase abolished the DNA relaxing activity of DNA topoisomerase I and the sensitivity of the enzyme to its specific inhibitor, camptothecin. DNA topoisomerase I could be reactivated by incubation with purified protein kinase C. DNA topoisomerase I was then able to relax supercoiled DNA processively, like the native enzyme, and to cleave 32P-end-labeled SV40 DNA fragments at the same sequences as the native enzyme in the presence of camptothecin. These results show that active DNA topoisomerase I is a phosphoprotein and suggest a possible regulatory role of protein kinase on topoisomerase I activity and on its sensitivity to camptothecin.

Binding of adenosine diphosphoribosyltransferase to the termini and internal regions of linear DNAs

Binding mechanisms of ADPR-transferase to restricted double-stranded DNA fragments of SV40 and pBR322 DNA were determined by nuclease protection techniques. Top and bottom strands of double-stranded DNA were identified by specific labeling with 32P. Protection against specific exonucleases identified binding of ADPR-transferase to DNA termini, whereas binding to internal regions of linear DNAs was probed by protection against endonucleases. ADPR-transferase protein protected against exonucleolytic attack from lambda exo and exoIII in all DNA fragments tested, demonstrating that the enzyme protein binds indiscriminately to all DNA termini. Extending earlier results [Sastry, S.S., & Kun, E. (1988) J. Biol. Chem. 263, 1505-1512], the modifying effect of the binding of ADPR-transferase to DNA induced changes in DNA conformation, as evident from altered pause sites that appeared following digestion of DNA fragments by lambda exonuclease in the presence of ADPR-transferase. In contrast to the nonselective binding of ADPR-transferase to DNA termini, ADPR-transferase conferred protection endonuclease attack (DNase I and micrococcal nuclease) only to the 209-bp EcoRI-PstI SV40 DNA fragment. These results indicate that binding of ADPR-transferase to relatively rare internal regions of restricted DNA fragments exhibits some degree of specificity. Specificity of binding appears to be related to the coincidental relative A+T-rich regions in DNA, and to DNA bending, both identified in the 209-bp SV40 DNA fragment. Synthetic polydeoxyribonucleotides containing dA-dT bind ADPR-transferase stronger than polydeoxyribonucleotides containing dG-dC. It was deduced from endonuclease protection patterns that binding of the enzyme protein leaves no defined footprints on the 209-bp SV40 DNA fragment, but there is significant modification of DNA structure following binding of the enzyme protein. Methylation protection assays and the prevention of the binding of ADPR-transferase to T4 DNA by its glucosylation indicate that the enzyme binds in the major groove of DNA. The 36-kDa A peptide fragment of ADPR-transferase [Buki, K. G., & Kun, E. (1988) Biochemistry 27, 5990-5995] exhibits the same protection against endonucleolytic enzymes as the intact ADPR-transferase molecule.

Homologous recombination of SV40 DNA in COS7 cells occurs with high frequency in a gene dose independent fashion.

Homologous recombination between microinjected SV40 DNA fragments and endogenous SV40 DNA in COS7 cells was analysed by immunofluorescence staining and DNA blotting. Time course experiments revealed that recombination between the transferred (trans) DNA and the chromosomal DNA occurred about 8 hours after microinjection with high efficiency in a gene dose independent fashion. Deletions of up to 1018 basepairs (bp) within the early or the late SV40 region were efficiently repaired after the transfer of linear but not of circular DNA molecules. A 22 bp homology between the trans DNA and the endogenous DNA was sufficient to initiate recombination but 14 nonhomologous bp at one open end of the SV40 DNA fragments hindered gap repair.

Molecular interactions between DNA, poly(ADP-ribose) polymerase, and histones.

Molecular interactions between purified poly(ADP-ribose) polymerase, whole thymus histones, histone H1, rat fibroblast genomic DNA, and closed circular and linearized SV40 DNA were determined by the nitrocellulose filter binding technique. Binding of the polymerase protein or histones to DNA was augmented greatly when both the enzyme protein and histones were present simultaneously. The polymerase protein also associated with histones in the absence of DNA. The cooperative or promoted binding of histones and the enzyme to relaxed covalently closed circular SV40 DNA was greater than the binding to the linearized form. Binding of the polymerase to SV40 DNA fragments in the presence of increasing concentrations of NaCl indicated a preferential binding to two restriction fragments as compared to the others. Polymerase binding to covalently closed relaxed SV40 DNA resulted in the induction of superhelicity. The simultaneous influence of the polymerase and histones on DNA topology were more than additive. Topological constraints on DNA induced by poly(ADP-ribose) polymerase were abolished by auto ADP-ribosylation of the enzyme. Benzamide, by inhibiting poly(ADP-ribosylation), reestablished the effect of the polymerase protein on DNA topology. Polymerase binding to in vitro-assembled core particle-like nucleosomes was also demonstrated.

The AT-rich sequence of the SV40 control region influences the binding of SV40 T antigen to binding sites II and III

During lytic infection SV40 T antigen binds specifically to three different regions of the SV40 DNA to initiate DNA replication and to regulate early and late transcription. We constructed plasmids containing either 23-bp synthetic oligonucleotides representing site I or II or SV40 DNA fragments with combinations of binding sites II and III with or without SV40 specific flanking regions. These plasmids were used to determine which sequences are sufficient for specific binding to isolated regions II and III. Under identical conditions T antigen bound in a sensitive in vitro binding assay efficiently to site I but not to the corresponding oligonucleotide of site II. Binding to site II could only be observed in the presence of the adjacent 17-bp AT-rich region of the SV40 DNA. On account of the markedly low affinity for binding site II, T antigen concentrations were required which exceeded those necessary to achieve saturation of binding to site I. The very low affinity for isolated site III could be slightly raised by the same AT-rich region. An increased binding to site II at 37° compared to 0° in the presence of this region points to an indirect influence on the DNA structure of the binding site.

Unique positioning of reconstituted nucleosomes occurs in one region of simian virus 40 DNA.

A direct end label method was used to study the positioning of nucleosome arrays on several long (greater than 2200 base pairs) SV40 DNA fragments reconstituted in vitro with core histones. Comparison of micrococcal nuclease cutting sites in reconstituted and naked DNA fragments revealed substantial differences in one DNA region. When sufficient core histones were annealed with the DNA to form closely spaced nucleosomes over most of the molecule, a uniquely positioned array of four nucleosomes could be assigned, by strict criteria, to a 610-base pair portion of the SV40 "late region," with a precision of about +/- 20 base pairs. In some other DNA regions, a number of alternative nucleosome positions were indicated. The uniquely positioned four-nucleosome array spanned the same 610 nucleotides on two different DNA fragments that possessed different ends. Removal of a DNA region that had contained a terminal nucleosome of the array, by truncation of the fragment before reconstitution, did not affect the positioning of the other three nucleosomes. As the core histone to DNA ratio was lowered, evidence for specific positioning of nucleosomes diminished, except within the region where the four uniquely positioned nucleosomes formed. This region, however, does not appear to have a higher affinity for core histones than other regions of the DNA.

Distribution of ultraviolet-induced lesions in simian virus 40 DNA

In order to analyze the molecular mechanisms of mutagenesis in mammalian cells, we devised an analytical assay using Simian Virus 40 as biological probe. To study the possible correlations between the distribution of the lesions on the treated DNA and the distribution of mutations, we have located and quantified the lesions induced by ultraviolet light (254 nm) on a SV40 DNA fragment. At a fluence of 2,000 J/m2, our results show that the formation frequency of thymine-thymine dimers (TT) is three to four times higher than the formation frequency of the other types of dimers (TC, CT, CC). On the other hand, the formation frequency of a dimer is influenced by the adjacent sequence. In particular, a pyrimidine in the 5' position of a thymine-thymine dimer enhances its formation frequency. At the dose used the formation frequency of the pyrimidine (6-4) pyrimidone photoproducts is twenty times less than the formation frequency of pyrimidine dimers. This paper shows the distribution of the major lesions induced by UV-light on a defined fragment of SV40 genome after UV irradiation. This work is necessary to get an insight into the molecular mechanisms of UV-mutagenesis.

Detection of an element of the SV40 late promoter in vectors used for expression studies in COS cells.

Plasmids containing hepatitis B virus (HBV) DNA and a 232-bp SV40 DNA fragment encoding the origin of replication were constructed. When introduced by transfection into COS cells, these plasmids directed the synthesis of hepatitis B surface antigen. S1 mapping of the mRNAs covering the S gene showed that transcriptional initiation was promoted by the interaction of HBV sequences with an SV40 promoter element: transcription started on HBV DNA but had several properties of SV40 late transcription. The detection of a promoter element in an SV40 origin fragment commonly used in the COS system is important for the interpretation of data deriving from expression studies in COS cells.

A specific replication origin in the chromosomal rDNA of Lytechinus variegatus.

Specific replication origins have been well characterized in prokaryotes1,2, and have been identified in eukaryotic extra-chromosomal DNAs3–8. The best candidate, until now, for a specific eukaryotic chromosomal replication origin has been found in yeast9, but the general existence of such origins is challenged10 by the finding that SV40 and polyoma DNA fragments lacking the viral replication origin can replicate after injection into Xenopus eggs. It has thus been suggested that the origins found on extrachromosomal DNAs exist solely to circumvent the cells' strict requirement for chromosomal DNA to replicate once per cell division. Clearly the disparate views can only be reconciled after the replication of specific chromosomal genes has been studied. To this end, we have studied the replication of the ribosomal genes (rDNA) of the sea urchin Lytechinus variegatus. The visualization of replicating rDNA, isolated from rapidly dividing sea urchin gastrula cells, after restriction endonuclease digestion demonstrates that the initiation of replication in these chromosomal genes is sequence-specific, and is most probably confined to a region within the non-transcribed spacer.

Formation of a nucleosome-free region in SV40 minichromosomes is dependent upon a restricted segment of DNA

We have analyzed minichromosomes of SV40 mutants with the electron microscope. In two insertion mutants, containing the SV40 DNA fragment from 0.66 to 0.715 map unit duplicated diametrically opposite to its original site in two alternative orientations, one nucleosome-free region (“gap”) was visualized in 25% of the molecules of each mutant with equal frequency either at the original position or at the insertion point. In a host-substituted mutant (F161F), where the viral DNA sequences spanning 0.67–0.73 map unit are interspersed as four copies between cellular sequences, one gap was visualized in almost 100% of the molecules in the vicinity of the viral DNA sequences. These observations indicate that gap formation is dependent upon SV40 DNA sequences located between 0.67 and 0.715 map unit.

Expression of herpesvirus thymidine kinase gene under control of early promoter of SV40

A 0.31-kilobase fragment of SV40 DNA containing the early promoter of this virus was isolated. The pX1 plasmid containing the herpesvirus thymidine kinase gene (TK) was cleaved with BglII and SalI to remove the TK promoter. The Sv40 DNA fragment was mixed with the TK gene without its promoter and blunt-end ligated after treatment with S 1 nuclease. The newly engineered plasmid was used for the transformation of TK-deficient LtA cells to TK + phenotype. Blot hybridization experiments show that the plasmid is integrated into high-molecular-weight DNA in both of the transformed colonies that we have studied. Also the mRNA in these cells is a hybrid molecule containing both TK and SV40 sequences.

Alternative interactions of the SV40 a protein with DNA

We have characterized the DNA binding properties of SV40 A protein (T antigen) from permissive cells. The viral protein protects 110- to 115-, 60- to 65-, 40- to 45-, and 30- to 35-bp fragments of SV40 DNA from an excess of DNase 1. The 60- to 65-bp fragment extends some 30–35 by to either side of the BglI site at the origin of replication. DNase footprinting confirms the location of this binding region and shows that it is the most completely protected DNA binding site under our conditions. The A protein selectively recognizes the 60- to 65-bp origin fragment in the complete absence of adjacent DNA and binds to it in three alternative ways that protect 60–65, 40–45, or 30–35 by from nuclease. Thus these three interactions with isolated origins recapitulate the binding of viral protein to intact SV40 DNA. We propose that the alternative interactions of SV40 A protein with the 60- to 65-bp origin fragment may represent three different conformational alignments of protein with DNA rather than a simple repetition of a single kind of binding interaction. Viral protein also protects pBR322 and λ phage DNA from nuclease to a limited extent. Similar interactions could possibly alter the function of DNA control regions of host cells.

Growth potential of sheep and sea mammal cells transformed by SV40 early region DNA.

AbstractNormal fibroblasts of bearded seal, northern fur seal, sea lion, domestic sheep, and normal kidney cells of spotted dolphin and Pacific common dolphin have been transformed by fragments of SV40 DNA containing the entire early region. The transformed, T-antigen-positive cell strains grew to higher saturation densities and, except for the transformed dolphin kidney cell strains, were able, albeit poorly, to form colonies in soft agar medium. The transformed northern fur seal, spotted dolphin and Pacific common dolphin cell strains have remained diploid, the transformed sheep cells have become hypodiploid due to Robertsonian translocations, and the transformed bearded seal and sea lion cells had become hypotetraploid. Although all the transformed cells have acquired increased divisional potential and prolonged lifespan in cell culture, only the hypotetraploid cell strains of bearded seal and sea lion have evolved into continuous cell strains.

74] Microinjection of early SV40 DNA fragments and T antigen

All early SV40 functions tested so far (Table I) are also detectable iin cells microinjected with the SV40 DNA fragment HpaII/BamHI A (map coordinates 0.16–0.735, Fig. 4). By contrast, fragments cut upstream of map position 0.16 have lost the ability to induce late gene expression (complementation of tsA virus at 41.5°), U antigen synthesis, or helper function for adenovirus 2. However, fragment BumI A, containing about 70% of the early coding region, still induces cellular DNA synthesis, whereas of all the functions tested, only intranuclear T antigen could be detected upon microinjection of the HpaII/HpaI B fragment.17,18 DNA sequences located at, or clockwise beyond the Bg/I cut (map position 0.67) seem to be required for correct intranuclear transcription, since the fragment Bg/I/BamHI failed to induce early SV40 functions in microinjected cells (Table I).17 However, cRNA transcribed from this fragment is translated into a protein(s) with SV40 T and U antigen specificity and the capacity to stimulate cell DNA synthesis following microinjection.

Monkey cells transformed by SV40 DNA fragments: flat revertants synthesize large and small T antigens.

SV40 causes tumor formation in animals and transforms tissue-culture cells. Experiments with early temperature-sensitive mutants (tsA) indicated that synthesis of wild-type (wt) SV40 large T antigen is required for the induction and maintenance of the transformed state (Martin and Chou 1975; Tegtmeyer 1975; Brugge and Butel 1975; Osborn and Weber 1975). Also, SV40 small T antigen seems to be involved in the process of cell transformation. Early SV40 mutants with deletions between 0.54 and 0.59 map units (e.g. dl884) code for wild-type large T antigen, but for small T antigens of reduced sizes (Bouck et al. 1978; Crawford et al. 1978; Sleigh et al. 1978). These mutants have reduced transformation capacities. However, it is still uncertain which of the early SV40-specific functions attributed to the viral T antigens determine the oncogenicity of SV40.