Sequence requirements for plasmid nuclear import

The nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of nonviral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366-bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72-bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in nonviral vectors may greatly increase their ability to be transported into the nucleus, especially in nondividing cells.     

Activation of the human epsilon- and beta-globin promoters by SV40 T antigen.

We have studied the effect of the SV40 T antigen on expression from human globin promoters fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and compared its effect with the SV40 enhancer and the adenovirus E1A protein. We have observed that expression of p epsilon GLCAT and p beta GLCAT (the epsilon-globin or beta-globin promoter linked to the CAT gene) was significantly stimulated when cotransfected with a cloned T antigen plasmid into CV-1 cells, indicating that trans-activation of the globin promoters was mediated by SV40 T antigen. Transfection of the p beta GLCAT-SV (p beta GLCAT containing the SV40 enhancer element) into CV-1 cells resulted in a 50-60-fold increase in CAT activity as compared to p beta GLCAT (no enhancer). However, cotransfection of the p beta GLCAT-SV with the cloned T antigen resulted in an additional increase of CAT expression, which suggests that T antigen and the SV40 enhancer activate globin gene expression independently. We found that T antigen but not E1A could further stimulate the expression of an enhancer-containing plasmid in CV-1 cells; whereas E1A but not T antigen could further stimulate p epsilon GLCAT expression in COS-1 cells which constitutively express the SV40 T antigen. These results suggest that T antigen and E1A also act independently. Deletion analysis showed that the minimum sequence required for a detectable level of stimulation of the epsilon-globin promoter by T antigen is 177 bp 5' to the cap site, suggesting that the target sequences for response to T antigen do not reside in the canonical 100 bp promoter region, but rather reside in sequences further upstream, and therefore the cellular factors interacting with T antigen are not the TATA or CAT box binding proteins, but the proteins interacting with upstream regulatory sequences.     

Initiation of SV40 DNA replication after microinjection into Xenopus eggs

We have examined the capacity of Xenopus laevis eggs to support replication of microinjected SV40 DNA. As previously reported, microinjected DNA undergoes semi-conservative replication. Unlabeled SV40 DNA was microinjected with [³H]dTTP and, after a 3 h incubation period, the DNA was recovered and adsorbed to BND-cellulose. Elution with an NaCl gradient removes molecules that are entirely double-stranded but not those with single-stranded regions. The latter DNA population is eluted with caffeine. The radioactive DNA that eluted with NaCl was comprised mostly of supercoiled and open circular SV40 DNAs. The radioactive DNA eluted with caffeine was comprised mainly of endogenous DNA but also contained replicative forms of SV40 DNA. Analysis of SV40 DNA replication intermediates by electron microscopy revealed mainly Cairn's forms of varying degrees of maturation. Digestion with BamH1, which cleaves SV40 DNA almost opposite the normal viral replication origin, indicated that SV40 DNA microinjected into frog eggs does not initiate DNA synthesis at its normal initiation site nor at any other obvious preferred site. Rather, it appears that when this template is injected into activated Xenopus eggs, replication may initiate at random.     

Expression of antibody cDNA in murine myeloma cells: possible involvement of additional regulatory elements in transcription of immunoglobulin genes

Expression vectors for cDNA of the κ and λ1 chains of a monoclonal antibody directed against creatine kinase were introduced into murine myeloma cells. κ and γ1 cDNA were either under the control of the SV40 early promoter or of the cognate promoters and enhancers of the light- and heavy-chain genes. Secretion of immuno-reactive κ and γ1 chains into the culture medium was demonstrated with the SV40 promoter as well as with the cognate promoters. Expression of y 1 cDNA with the SV40 early promoter was about twice as high as with the heavy-chain promoter and enhancer. Expression of κ cDNA under the control of the S V40 early promoter was about 17 times higher than with the light-chain promoter and enhancer. These expression levels were compared to those of a genomic immunoglobulin (Ig) κ determinant, including introns. Such an entire κ gene led to expression of the light chain at levels double those with the κ cDNA construction using the SV40 promoter and about 35 times as high when using κ cDNA and the cognate promoter and enhancer. This result might indicate that, besides the cognate promoter and enhancer elements, other intragenic elements are involved in the regulation of Ig expression. However, the SV40 early promoter seems to be able to compensate for the absence of these postulated regulatory elements probably located in the introns.     

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.