Restriction enzyme mapping of vaccinia virus DNA.

The cleavage sites for the restriction enzymes Bg/I, HindIII, KpnI, SalI, SmaI, and XhoI were located, from primary data, on the DNA isolated from the WR strain of vaccinia virus. Bg/I and SmaI divide the DNA into five segments which can be isolated by sucrose gradient centrifugation. These large segments provide a convenient means to group segments produced by other enzymes. The construction of physical maps was initiated by identifying the segments at each end of the DNA and then finding segments which were adjacent to these terminal sections. This was accomplished by isolating large shear fragments which contained the covalently linked termini of the DNA. Most of the data needed to derive the maps were obtained by isolating segments produce by one enzyme and then cleaving these individual segments with a second enzyme.     

Generation of a telomere-based episomal vector

We have developed a telomere-based episome by large-scale amplification in Escherichia coli cells. This episome consists of a PAC vector in which a 6 Kb sequence, containing an array of telomeric repeats spaced by a synthetic sequence, is tandemly repeated by large-scale multimerization in E. coli. After transfection in human HT1080 cells, the construct, called clone 106, was able to persist in episomal form or integrated into some endogenous chromosomes. Integrations occurred exclusively at the telomeres. Episomes were still present in HT1080 cells after more than 100 days in the absence of selection. Integrations of clone 106 into the telomeric regions were retained only under selective conditions, and when the selection was removed the construct was progressively eliminated from the chromosome. The long-term maintenance of clone 106 into human cells as an episome and its ability to integrate transiently into the telomeres of the host chromosomes suggest that this PAC-based episome is potentially a good candidate vector for gene therapy applications.     

Restriction mapping of recombinant lambda DNA molecules using pulsed field gel electrophoresis

We have integrated pulsed field gel electrophoresis with the partial digestion strategy of Smith and Birnstiel (1976, Nucleic Acids Res. 3,2387-2398) to generate a rapid and accurate method of restriction endonuclease mapping recombinant lambda DNA molecules. Use of pulsed field gels dramatically improves the accuracy of size determination and resolution of DNA restriction fragments relative to standard agarose gels. Briefly, DNA is partially digested with restriction enzymes to varying extents and then hybridized with a radiolabeled oligonucleotide which anneals specifically to one of the lambda cohesive (cos) ends, effectively end labeling only those digestion products containing that cos end. In this study, we have used an oligonucleotide hybridizing to the right cos end. DNA is then fractionated by pulsed field gel electrophoresis, the gel dried down, and cos end containing fragments visualized by autoradiography. Fragment sizes indicate the distances from the labeled cos end to each restriction site for the particular restriction enzyme employed. This procedure requires only minimal quantities of DNA and is applicable to all vectors utilizing lambda cos ends.     

An episomal vector for stable tetracycline-regulated gene expression.

The recently introduced tetracycline (Tc)-regulatable eukaryotic gene expression system based on the Escherichia coli Tn 10 tetracycline operon has proven to be a powerful tool for controlled expression of a variety of genes in vitro as well as in vivo . Control elements of this expression system are contained in two separate plasmid vectors. The tTA vector encodes a transactivator protein and the tetP vector contains a responsive operator-promoter element (tetP) that controls gene expression depending on tTA binding. Establishment of cell lines expressing a gene of interest under tetP control requires two subsequent rounds of transfection and clonal selection after each transfection. Here we describe a modification of this system in which the tetP element is placed in an episomal EBNA-based plasmid that can be stably maintained in primate but not in rodent cells. Using HeLa and human melanoma cells, we show that upon transient or stable transfection a reporter gene is expressed in a Tc-regulated manner similar to the original system. Thus, this expression system combines the advantages of episomal vectors, such as high efficiency of transfection and time-efficient selection of mass cultures, with tight control of gene expression provided by the Tc-regulatable system.     

Restriction factors: a defense against retroviral infection

Susceptibility to retroviral infection is determined, in part, by host genes with antiviral activity. The Fv1 gene, which inhibits murine leukemia virus infection in mice, encodes one such resistance factor, and was long thought to be unique in that it restricts post-entry, pre-integration steps of retroviral replication. However, recent findings suggest the existence of similar restriction factors in primates, including humans. These factors, termed Lv1 and Ref1, can inhibit a range of retroviruses, including human immunodeficiency virus type 1 and its relatives. Fv1, Lv1 and Ref1 target capsid determinants to block infection but can be saturated by incoming virions. Primate- and murine-retrovirus restriction factors have diverse and overlapping specificities, and some variants of Lv1, as well as Ref1, apparently recognize and inhibit infection by widely divergent retroviruses.     

Inducible microRNA expression by an all-in-one episomal vector system

Here we describe an episomal, one-vector system which allows the generation of cell populations displaying homogenous, inducible gene inactivation by RNA interference in a one step procedure. A dual tet-repressor/activator system tightly controls a bi-directional promoter, which simultaneously drives expression of microRNAs and a fluorescent marker protein. We demonstrate the effectiveness of this vector by knockdown of p53 expression in a human cell line which resulted in the expected loss of G₁-arrest after DNA damage. The generation of a cell pool homogenously expressing the ectopic microRNAs was achieved in 1 week without the need for viral infections. Induction of microRNA expression did not elicit an interferon response. Furthermore, the vector was adapted for convenient ligation-free transfer of microRNA cassettes from public libraries. This conditional knockdown-system should prove useful for many research and gene therapeutic applications.     

Restriction endonuclease mapping of linear unintegrated proviral DNA of bovine leukemia virus.

A detailed restriction map was deduced for the genome of the exogenous bovine leukemia virus. The cleavage sites for nine restriction enzymes were mapped. The unintegrated linear viral DNA intermediate that is produced by infection of permissive cells with bovine leukemia virus was isolated. The linear viral DNA had a unique restriction map, indicating that it is not a set of random circular permutations of the RNA genome. From hybridization with a 3'-enriched probe, the DNA restriction map was aligned relative to the 5'-to-3' orientation of the viral RNA. Restriction enzyme analysis of integrated bovine leukemia virus information present in animals with enzootic bovine leukosis provided evidence for the existence of genetic variants of the virus.     

An S/MAR-based infectious episomal genomic DNA expression vector provides long-term regulated functional complementation of LDLR deficiency

Episomal gene expression vectors offer a safe and attractive alternative to integrating vectors. Here we describe the development of a high capacity episomal vector system exploiting human episomal retention sequences to provide efficient vector maintenance and regulated gene expression through the delivery of a genomic DNA locus. The iBAC-S/MAR vector is capable of the infectious delivery and retention of large genomic DNA transgenes by exploiting the high transgene capacity of herpes simplex virus type 1 (HSV-1) and the episomal retention properties of the scaffold/matrix attachment region (S/MAR). The iBAC-S/MAR vector was used to deliver and maintain a 135kb genomic DNA insert carrying the human low density lipoprotein receptor (LDLR) genomic DNA locus at high efficiency in CHO ldlr^−/− a7 cells. Long-term studies on CHO ldlr^−/− a7 clonal cell lines carrying iBAC-S/MAR-LDLR demonstrated low copy episomal stability of the vector for >100 cell generations without selection. Expression studies demonstrated that iBAC-S/MAR-LDLR completely restored LDLR function in CHO ldlr^−/− a7 cells to physiological levels and that this expression can be repressed by ~70% by high sterol levels, recapitulating the same feedback regulation seen at the endogenous LDLR locus. This vector overcomes the major problems of vector integration and unregulated transgene expression.     

The control region of the F sex factor DNA transfer cistrons: Restriction mapping and DNA cloning

Summary A restriction endonuclease map of EcoRI fragment f6 of F sex factor DNA was constructed and aligned with pre-existing physical and genetic maps. Results of genetic complementation tests and analysis of proteins synthesized in minicells from PstI and BglI₁ sub-fragment clones, or from a specific BglII fragment deletion, have allowed mapping of the locations of the origin of DNA transfer and many of the transfer genes known to lie on f6. The proteins detected account for 78% of the coding capacity of fragment f6.     

Restriction mapping and molecular cloning of adenovirus type 4 (subgroup E) DNA

The locations of thirty restriction endonuclease cleavage sites were determined on the genome of adenovirus type 4 (Ad4), the sole member of the subgroup E adenovirions. The restriction endonucleases BglII, EcoRI, HindIII, HpaI, KpnI, SalI, and XbaI cut Ad4 DNA 10, 3, 2, 3, 5, 5 and 3 times, respectively. Orientation of the linear Ad4 map with respect to left and right molecular ends was accomplished by taking advantage of the limited sequence homology between Ad2 and Ad4. Ten non-overlapping fragments of Ad4 DNA representing 98% of the genome, map units 1.6 to 99.6, have been cloned into the plasmid vector pKC7.     

Enhancing expression level and stability of transgene mediated by episomal vector via buffering DNA methyltransferase in transfected CHO cells

Nonviral episomal vectors present attractive alternative vehicles for gene therapy applications. Previously, we have established a new type of nonviral episomal vector-mediated by the characteristic motifs of matrix attachment regions (MARs), which is driven by the cytomegalovirus (CMV) promoter. However, the CMV promoter is intrinsically susceptible to silencing, resulting in declined productivity during long-term culture. In this study, Chinese hamster ovary (CHO) cells and DNA methyltransferase-deficient (Dnmt3a-deficient) CHO cells were transfected with plasmid-mediated by MAR, or CHO cells were treated with the DNA methylation inhibitor 5-Aza-2′-deoxycytidine. Flow cytometry, plasmid rescue experiments, fluorescence in-situ hybridization (FISH), and bisulfite sequencing were performed to observe transgene expression, its state of existence, and the CpG methylation level of the CMV promoter. The results indicated that all DNA methylation inhibitor and methyltransferase deficient cells could increase transgene expression levels and stability in the presence or absence of selection pressure after a 60-generation culture. Plasmid rescue assay and FISH analysis showed that the vector still existed episomally after long-time culture. Moreover, a relatively lower CMV promoter methylation level was observed in Dnmt3a-deficient cell lines and CHO cells treated with 5-Aza-2′-deoxycytidine. In addition, Dnmt3a-deficient cells were superior to the DNA methylation inhibitor treatment regarding the transgene expression and long-term stability. Our study provides the first evidence that lower DNA methyltransferase can enhance expression level and stability of transgenes mediated by episomal vectors in transfected CHO cells.     

GUS expression patterns from a tobacco yellow dwarf virus-based episomal vector

We have constructed a binary vector containing elements of the monopartite geminivirus, tobacco yellow dwarf virus (TYDV). The vector is designed to be stably integrated into the plant genome, via Agrobacterium-mediated transfer. Upon expression of the viral replication-associated protein the TYDV elements are released from the T-DNA and then replicate episomally. We refer to these released forms as multicopy plant episomes (MPE). Tobacco plants (Nicotiana tabacum cv `Samsun') transformed with the vector showed MPE release and subsequent episomal replication in 6 of 11 of these plants. An MPE vector containing the uidA gene faithfully replicated and maintained the reporter gene, even though the construct was considerably larger than the wild-type TYDV genome. Histochemical staining revealed a speckled GUS expression phenotype which could be correlated with MPE replication. Received: 11 July 1997 / Revision received: 4 November 1997 / Accepted: 8 December 1997     

Restriction enzyme mapping of the DNA of Streptomyces bacteriophage B alpha and its deletion derivatives

Cleavage analysis of actinophage B alpha DNA was done with several restriction enzymes, and a restriction map of the DNA was determined. The DNA appeared to carry cohesive ends. Deletion mutants of actinophage B alpha were isolated by five cycles of treatment with 15 mM PPi. Both mutants had deletions of 2.5 of 1.8 megadaltons near one end of the genome, and one of them lost the single EcoRI cleavage site.