Aspects of cellular physiology that influence DNA-mediate gene transfer in NIH3T3 cells.

Cellular physiology has a significant influence on the efficiency of various gene transfer procedures, as shown by the fact that transfection efficiency varies dramatically among different cell lines. However, the aspects of cellular physiology which influence the transfection process remain substantially uncharacterized. In this study, NIH3T3 cells were treated with inhibitors of protein synthesis, DNA synthesis, and RNA synthesis to determine the importance of these processes in the calcium-phosphate transfection process. The results suggest that protein synthesis during the first 4 h after DNA addition enhances transfection. In contrast, inhibition of RNA synthesis has no effect on transfection during the first 24 h post-DNA addition. The DNA synthesis inhibitor results remain inconclusive due to a secondary inhibition of an unknown cellular factor. Secondly, agents that destabilize microtubules, microfilaments, and the golgi apparatus were used to determine whether these elements play a role in the transfection process. The results suggest that microtubules are not involved in the transfection process, microfilaments are important but not necessary for the transfection process, and a functional golgi apparatus is essential early in the transfection process. These studies provide a foundation from which further investigations into the cellular processes involved in the uptake and expression of exogenous DNA can proceed.

Isolation and expression of rat thymidylate synthase cDNA: phylogenetic comparison with human and mouse thymidylate synthases.

Two cDNA clones representing rat hepatoma thymidylate synthase (rTS) were isolated from a lambda ZAP II cDNA library using as a probe a fragment of the human TS cDNA. The two were identical except that one was missing 50 bp and the other 23 bp corresponding to the 5' coding region of the protein. The missing region was obtained by screening a rat genomic library. The open reading frame of rTS cDNA encoded 921 bp encompassing a protein of 307 amino acids with a calculated molecular mass of 35,015 Da. Rat hepatoma TS appears identical to normal rat thymus TS and the two sequences differ from mouse TS in the same eight amino acid residues. Six of these differences are in the first 21 amino acids from the amino-end. The human enzyme differed from rat and mouse TS at 17 residues where the latter two were identical, with most changes being conservative in nature. The three species differed completely at only four sites. Because the mouse TS shares four amino acids with human TS at sites which differ from rTS and a comparable situation does not exist between rTS and human TS, it is suggested that mouse TS is closer to human TS phylogenetically than rTS. The polymerase chain reaction was used to subclone the protein coding region of rTS into a high expression vector, which expressed rTS in Escherichia coli to the extent of 10 to 20% of its cellular protein. Although the amino-end of the amplified TS was unblocked, that isolated from a FUdR-resistant rat hepatoma cell line contained mostly N-acetylmethionine on its N-terminal end, a finding that may have significant regulatory consequences, which are discussed. The TS level in the resistant cell line was 60 to 70-fold higher than normal which was found to be associated with both multiple gene copies and an expanded TS mRNA pool.

Structure of the terminal 300 kb of DNA from human chromosome 21q.

The most distal 300 kb of human chromosome 21q was cloned and mapped using telomeric yeast artificial chromosomes (YACs). The region contains low-copy subtelomeric repeats at the telomeric end, chromosome 21-specific sequences more centromerically, and the S100B gene at a distance of 100-140 kb from the chromosome terminus. RecA-assisted restriction endonuclease cleavage of genomic DNA showed that the cloned fragments correspond to telomere-terminal genomic DNA, and restriction enzyme mapping of the YACs shows that the smaller clone (175 kb) corresponds exactly to the telomeric end of the larger one (300 kb). PCR assays for 21q-specific markers were used to show that COL6A1, COL6A2, and LA161 were all outside of the subtelomeric region spanned by the YACs and thus at least 300 kb from the 21q terminus. The molecular probes provide telomeric closure for existing 21q maps.

Differential effects of the phorbol ester TPA on DNA-mediated transfection in a variety of cell lines.

The efficiency of stable gene transfer and expression in NIH3T3 cells has been shown to be significantly enhanced by a brief treatment with the phorbol ester tetradecanoylphorbol 12,13-acetate (TPA) immediately following calcium-phosphate transfection. Several lines of evidence indicated that this effect was mediated through protein kinase C activation. These studies were expanded to determine whether this was a consistent and widespread phenomenon among other cell lines. The efficiency of transfection in two other established fibroblast lines, LMtk- and 2A3 3T3, was unaffected by TPA treatment, and primary human foreskin fibroblasts were similarly unaffected. Transfection was inhibited by TPA treatment in the transformed cell lines EJ and HeLa. Protein kinase C enzyme assays indicated that TPA causes a translocation of the enzyme from cytosol to membrane in both NIH3T3 and EJ cells, suggesting that the PKC translocation event does not account for the TPA effect on transfection. The TPA-mediated inhibition of transfection in EJ cells was not blocked by sphingosine, suggesting that this phenomenon is unrelated to PKC activation. The results suggest that TPA treatment may either enhance, inhibit, or have no effect on transfection, depending on the cell line.

Potentiation of DNA mediated gene transfer in NIH3T3 cells by activators of protein kinase C.

The mechanisms involved in the translocation of exogenously added genetic information through the cellular cytoplasm and into the nucleus are essentially unknown. Several trans-cytoplasmic translocation systems operate within cells to transport information received by the plasma membrane into the nucleus. Protein kinase C may be functionally involved in many of these translocation mechanisms. In order to explore the involvement of protein kinase C activation in the cytoplasmic translocation of DNA, NIH3T3 fibroblasts were transfected using the calcium-phosphate co-precipitation method with a plasmid containing the lacZ gene and treated with tetradecanoylphorbol 12,13-acetate (TPA) or 1,2-dioctanoylglycerol (DiC8). Addition of TPA or DiC8 immediately after glycerol shock resulted in a 5-7-fold increase in the number of cells expressing beta-galactosidase as well as a concomitant increase in the total amount of beta-galactosidase activity in the population during periods of transient and stable expression. TPA added at later times resulted in lesser increases in the efficiency of transfection. In contrast, TPA added at the time of addition of the calcium-phosphate precipitate inhibited transfection. In support of a role for protein kinase C activation in enhancing DNA transfection, the TPA analog 4 alpha-phorbol 12,13-didecanoate, which does not activate protein kinase C, was ineffective at enhancing transfection. Furthermore, treatment of cells with the protein kinase C inhibitor sphingosine blocked the TPA-mediated increase in transient and stable expression. The results suggest that protein kinase C activation enhances transfection of exogenous DNA through an as yet unknown mechanism.