We mapped polyoma virus-specific mRNAs isolated from productively infected mouse 3T6 cells on the viral genome by analyzing nuclease S1-resistant RNA-DNA hybrids. The polyoma early mRNAs, which code for the three T antigens, have several 5' ends near 73 map units (m.u.). During the late phase of infection an additional 5' end is found near 71 m.u. All of the major early mRNAs have common 3' ends at 26.01 m.u. There is a minor species of early mRNA with a 3' end at 99.05 m.u. There are two proximal and two distal splice junctions in the early region which are used to generate three different spliced early mRNAs. There are three late mRNAs encoding the three virion proteins, VP1, VP2, and VP3. The late mRNAs have common 3' ends at 25.34 m.u. The late mRNAs have heterogeneous 5' leader sequences derived from the region between 65.53 and 68.42 m.u. The leader sequences are joined to the bodies of the messages coding for VP2, VP3, and VP1 at 66.59, 59.62, and 48.57 m.u., respectively. These results confirm and extend previous analyses of the fine structure of polyoma mRNAs.
SUMMARY We constructed a recombinant DNA gene containing sequences from the 5′ flanking region of a dexamethasone-inducible rat growth hormone gene linked to the coding region of the polyoma virus transforming protein, the middle-size T (MT) antigen. We used this gene to derive cell lines in which the expression of the MT antigen could be regulated by dexamethasone. We transfected mouse NIH 3T3 cells and isolated transformed foci from cultures grown in the presence or absence of dexamethasone. The frequency of focus formation and the size of the transformed foci were increased in the presence of dexamethasone. Several transformants showed regulated expression of the MT antigen: the levels of polyoma-specific RNA, MT protein and MT-associated kinase activity were increased two- to fivefold in cells grown in the presence of dexamethasone. These results show that 248 base pairs of rat growth hormone DNA, including the first 237 base pairs upstream from the major transcription initiation site, contain promoter activity and a regulatory element required for glucocorticoid induction. This region of the rat growth hormone can be used to regulate expression of the polyoma MT antigen gene. In some cell lines regulated expression of the MT antigen was accompanied by regulated expression of transformed cell growth properties. The minimum level of the MT antigen required for expression of transformation was considerably less than the level found in a polyoma MT-transformed cell line. Increasing the level of the MT antigen led to increased expression of transformation, assayed by morphology, focus formation and growth in agar.
Electroporation is a process in which a controlled electrical pulse is applied to cells, inducing a transient destabilization of the cell membrane. During this time, the cells are highly permeable to exogenous substances in the surrounding media. DNA, proteins, and small molecules are all taken up by cells during electroporation; introduction of DNA into cells is the most common application. Gene transfer by electroporation offers many advantages for analysis of gene expression. The technique is simple, rapid, and reproducible. It is especially suited to suspension cultures and certain cell types that are poorly trans-fected by other means. Because all cells are transfected instantaneously, and essentially simultaneously, it is particularly suited to quantitation of gene transfer. Finally, single-copy, stable transfectants can often be isolated (1). Whereas the basic mechanisms of electroporation are still largely unknown, optimizing the conditions for electroporation of any particular cell type is primarily empirical.
INTRODUCTION Biolistics ("biological ballistics") or particle bombardment provides a rapid and simple physical procedure for delivering genes into cells (Klein et al. 1987; Yang et al. 1990). The technique has many advantages-plasmids may be used for delivery, DNA theoretically can be delivered to any cell type, and genes may be delivered to cells in vitro, ex vivo, or in vivo. DNA-coated gold particles are distributed evenly along the length of the tubing, which is subsequently cut into short sections of cartridges to be used in a gene gun. The Helios Gene Gun uses a pulse of helium to launch the DNA-coated particles, spreading them onto the target cells.
A rapid method for electrophoresis of DNA-protein complexes is described. Popular "gel-shift" assays are performed using Pharmacia PhastSystem with its convenience of pre-cast gels and buffer strips and microprocessor-controlled high-resolution separation. Using this system, the products of a DNA binding reaction (DNA-protein complexes) can be separated from "free" DNA in less than one hour. DNA fragments as well as oligonucleotides have been used as targets with partially purified extracts containing sequence-specific DNA binding proteins. We present here a comparison of results of gel-shift assays obtained by conventional techniques and by our rapid "PhastShift" method which reduces the time, effort and technical expertise necessary to obtain reproducible results.
