DNA-mediated gene transfer without carrier DNA.

DNA-mediated gene transfer is a procedure which uses purified DNA to introduce new genetic elements into cells in culture. The standard DNA-mediated gene transfer procedure involves the use of whole cell DNA as carrier DNA for the transfer. We have modified the standard DNA-mediated gene transfer procedure to transfer the Herpes simplex virus type 1 thymidine kinase gene (TK) into TK- murine recipient cells in the absence of whole cell carrier DNA. The majority (8/10) of carrier-free transformant lines expressed the TK+ phenotype stably, in sharp contrast to our results with carrier-containing DNA-mediated gene transfer. There was a wide range in donor DNA content among independent transformants. Further analysis on one transformant line using DNA restriction digests and in situ hybridization provided evidence that, in the absence of whole cell carrier DNA, multiple donor DNA sequences became integrated at a single chromosomal site.

Deposits of amyloid beta protein in the central nervous system of transgenic mice.

Alzheimer's disease is characterized by widespread deposition of amyloid in the central nervous system. The 4-kilodalton amyloid beta protein is derived from a larger amyloid precursor protein and forms amyloid deposits in the brain by an unknown pathological mechanism. Except for aged nonhuman primates, there is no animal model for Alzheimer's disease. Transgenic mice expressing amyloid beta protein in the brain could provide such a model. To investigate this possibility, the 4-kilodalton human amyloid beta protein was expressed under the control of the promoter of the human amyloid precursor protein in two lines of transgenic mice. Amyloid beta protein accumulated in the dendrites of some but not all hippocampal neurons in 1-year-old transgenic mice. Aggregates of the amyloid beta protein formed amyloid-like fibrils that are similar in appearance to those in the brains of patients with Alzheimer's disease.

In vitro methylation of bovine papillomavirus alters its ability to transform mouse cells.

Bovine papillomavirus (BPV) was methylated in vitro at either the 29 HpaII sites, the 27 HhaI sites, or both. Methylation of the HpaII sites reduced transformation by the virus two- to sixfold, while methylation at HhaI sites increased transformation two- to fourfold. DNA methylated at both HpaII and HhaI sites did not differ detectably from unmethylated DNA in its efficiency of transformation. These results indicate that specific methylation sites, rather than the absolute level of methylated cytosine residues, are important in determining the effects on transformation and that the negative effects of methylation at some sites can be compensated for by methylation at other sites. BPV molecules in cells transformed by methylated BPV DNA contained little or no methylation, indicating that the pattern of methylation was not faithfully retained in these extrachromosomally replicating molecules. Methylation at the HpaII sites (but not the HhaI sites) in the cloned BPV plasmid or in pBR322 also inhibited transformation of the plasmids into Escherichia coli HB101 cells.

Changes in structure and methylation pattern in a cluster of thymidine kinase genes.

Cell line 101 is a thymidine kinase (TK)-positive derivative of Ltk- which contains ca. 20 copies of the herpes simplex virus TK gene organized in a tandem array. DNA methylation at three sites within the gene and flanking sequences was inversely correlated with expression: the sites were unmethylated in line 101, methylated in each of 4 TK-negative derivatives of 101, and unmethylated in each of 21 TK-positive derivatives derived from them. The same three sites were affected in most of the 20 copies of the TK gene, whereas other sites between them were not affected. Although the entire gene cluster was never lost, indicating that integration into the genome was stable, internal rearrangements occurred at a high frequency. The rearrangements had no obvious correlation with the state of methylation or with the expression of the genes.

Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.

Deoxyribonucleic acid-mediated gene transfer in mammalian cells: molecular analysis of unstable transformants and their progression to stability.

To elucidate mechanisms involved in deoxyribonucleic acid-mediated gene transfer, we transferred the herpes simplex virus thymidine kinase gene (TK) into mouse Ltk- cells. Independent TK+ clones (transformants) and derivatives of each were tested for phenotypic expression and the presence and arrangement of TK sequences. Initially, transformants expressed viral TK unstable, with 10% of the cells in each generation losing both the TK+ phenotype and virally derived TK sequences. After a prolonged period in culture, stable subpopulations arose from which the TK+ phenotype and viral sequences were no longer lost at detectable frequency. Analysis of unstable cell populations indicated that individual viral deoxyribonucleic acid molecules were reduced in size, but were linked to other deoxyribonucleic acid to form molecules large enough to be precipitated in a Hirt fractionation. We term these molecules transgenomes. Analysis of independent unstable subclones derived from the primary transformants demonstrated that individual transgenomes could contain multiple copies of the viral TK sequences. Recipient cell lines frequently possessed more than one type of transgenome and possibly multiple copies per cell of each type. Stable derivatives possessed only one of the transgenomes present in the unstable parent, and these sequences were associated with a recipient cell chromosome.

Serum-free selection of onc genes.

We have described the development of a selective system useful for the identification and characterization of onc genes capable of conferring growth factor independence. The use of defined, serum-free media allows us to select for transformed cells which have lost specific growth factor requirements. We have used this system to show that several cloned onc genes generate different transformed phenotypes with respect to growth factor requirements. BPV-1 is active in relieving contact inhibition, yet these transformed NIH/3T3 cells retain their stringent requirement for FGF. In contrast, sis and H-ras were equally proficient at relieving contact inhibition and the requirement for FGF. Sis induced equal numbers of colonies regardless of the presence or absence of insulin, however, H-ras-mediated colony formation decreased four-fold when insulin was removed. This suggests that H-ras is less efficient in relieving the insulin requirement than is sis. To determine if colony formation by H-ras is a function of dosage, we are conducting experiments to measure the level of expression of p21 in transformants selected with and without insulin in the media. We have also presented data to show that loss of contact inhibition and loss of growth factor requirements are dissociable phenotypes under separate control in some cells. Thus, it should be possible to use this selective system to identify transforming genes in tumor DNA. Since some of these genes may be undetectable by the standard focus forming assay, selection in MSF medium may prove to be a useful tool for identifying and elucidating the action of activated cellular onc genes.

A unique pattern of toxic synthesis in pentitol catabolism: implications for evolution.

All of our Escherichia coli C mutants blocked in the first step of D-arabitol catabolism (D-arabitol dehydrogenase) became unable to grow in the presence of D-arabitol. We have shown that this sensitivity is eliminated by a defect in the second enzyme of the pathway (D-xylulokinase), leading to a pattern of toxicity and its relief which has not been previously reported. We have found a similar pattern of toxicity and its relief in the closely related ribitol pathway. The evolutionary significance of these findings is discussed.

Ribitol and D-arabitol catabolism in Escherichia coli.

In Escherichia coli C, the catabolism of the pentitols ribitol and D-arabitol proceeds through separate, inducible operons, each consisting of a dehydrogenase and a kinase. The ribitol operon is induced in response to ribulose, and the D-arabitol operon is induced in response to D-arabitol. Each operon is under negative control. The genes of the ribitol and D-arabitol operons are very closely linked and lie in a mirror image arrangement, rtlB-rtlA-rtlC-atlC-atlA-atlB, between metG and his on the E. coli chromosome.

Acquisition of ability to utilize Xylitol: disadvantages of a constitutive catabolic pathway in Escherichia coli.

Ribitol+ strains of Escherichia coli acquire the ability to utilize xylitol by mutating to constitutive production of the coordinately controlled ribitol catabolic enzymes ribitol dehydrogenase (RDH) and D-ribulokinase (DRK). Such strains concomitantly acquire toxicity to galacitol and L-arabitol, and to D-arabitol if they are unable to utilize it for growth. Strains selected for resistance to these polyols have DRK structural gene mutations or other mutations that eliminate the constitutive production of DRK, consistent with the view that DRK phosphorylates those polyols to toxic substances. Ribitol+ strains selected for growth on 8 mM xylitol fail to grow on 30 mM xylitol. A product of ribitol and xylitol catabolism represses synthesis of RDH, an enzyme required for growth on xylitol. At 30 mM xylitol, greater than 99% of RDH synthesis is repressed. Strains that grow on 8 mM xylitol can mutate to grow on 30 mM xylitol. Such mutants, relieved of this repression, overproduce RDH, resulting in good growth on the poor substrate, xylitol, but poor growth on the normal substrate, ribitol.

Cell type-specific expression of JC virus T antigen in primary and established cell lines from transgenic mice.

The highly restricted host range of JC virus (JCV) has made it difficult to study the biology of this common human papovavirus. To increase our understanding of the tissue specificity of this virus, we have examined the expression of the T antigen (T-Ag) in primary and established cell lines from various tissues of transgenic mice containing the JCV early region. In contrast to earlier results from a simian virus 40-containing transgenic mouse, there was no T-Ag expression in mesenchymal fibroblasts derived from two lines of JCV-transgenic mice. Instead, we isolated T-Ag-positive (T-Ag+) cells that had characteristics consistent with a neural crest origin. Furthermore, primary brain cultures contained many T-Ag+ astrocytes, but no expression was detected in macrophages, epithelial cells, neuronal cells nor, surprisingly, in oligodendrocytes. Continued passage of these cultures resulted in vigorously growing glial fibrillary acidic protein-positive, T-Ag+ astrocytes. Thus, the strict tissue specificity of JCV expression was maintained, despite the fact that the viral genome pre-existed in every tissue of these transgenic mice and these constraints on expression were preserved even when cells were explanted in vitro.

DNA-mediated gene transfer of a circular plasmid into murine cells.

We have used DNA-mediated gene transfer to introduce a recombinant plasmid containing the human beta-globin gene (H beta 1) into cells of a mouse tissue culture line, Ltk-. DNA isolated from independent transfer lines was analyzed by restriction endonuclease digestion, gel electrophoresis, modified Southern blotting, and filter hybridization using H beta 1 as a probe. H beta 1 sequences were present in 80% of the lines at 1-30 copies per cell. Many of the lines gave a hybridization pattern indicative of H beta 1 sequences integrated into high molecular weight DNA. DNA from three cell lines, digested with several restriction enzymes, produced a pattern providing evidence for the presence of circular H beta 1 molecules in the murine recipient cells.

Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes.

Coprecipitation of DNA with calcium phosphate is a commonly used method of gene transfer in mammalian cells. We have found that DNA forms a tight complex with Ca Pi and that DNA in this complex is resistant to nucleases present in serum or added externally. Under optimal conditions, virtually all of the recipient mouse Ltk- Aprt- cells take up Ca Pi--DNA complexes, as determined by fluorescent dyes specific for DNA (4',6-diaminilo-2-phenylindol dihydrochloride) or for calcium salts (chlorotetracycline). However, only a small proportion of the cells have detectable CA Pi--DNA complexes in the nucleus. Uptake of the Ca Pi--DNA complexes was highly dependent upon the pH at which the Ca Pi--DNA complexes was formed and upon the concentration of DNA in the complex.

Extinction of JC virus tumor-antigen expression in glial cell--fibroblast hybrids.

JC virus (JCV) is a ubiquitous human papovavirus that shares sequence and structural homology with simian virus 40 (SV40). In contrast to SV40, expression of JCV is restricted to a small number of cell types, including human fetal glial cells, uroepithelial cells, amnion cells, and some endothelial cells. To study the control of JCV early region expression, we made heterokaryons and stable hybrids between JCV-transformed hamster glial cells and mouse fibroblasts. Binucleate heterokaryons exhibited extinction of large tumor antigen expression in the hamster nuclei as assayed by indirect immunofluorescence. This extinction was both time and dose dependent: extinction reached maximal levels at 24-36 hr after fusion and was dependent on the ratio of glial cell to fibroblast nuclei in multinucleated heterokaryons. Extinction also was observed in stable hybrids between the glial cells and mouse Ltk- cells. Southern blot analysis showed that the extinguished hybrids contained viral sequences. Reexpression of large tumor antigen was observed in several subclones, suggesting that extinction was correlated with the loss of murine fibroblast chromosomes from these hybrids. The cis-acting region that mediates extinction resides within the viral regulatory region, which contains two 98-base-pair repeats that have enhancer activity. These data demonstrate that cellular factors that negatively regulate viral gene expression contribute to the restricted cell-type specificity of this virus.

Early regions of JC virus and BK virus induce distinct and tissue-specific tumors in transgenic mice.

JC virus and BK virus are ubiquitous human viruses that share sequence and structural homology with simian virus 40. To characterize tissue-specific expression of these viruses and to establish model systems for the study of human viral-induced disease, transgenic mice containing early regions of each of the viruses were produced. The viral sequences induced tumors in a distinct and tissue-specific manner that was similar to their tissue tropism in humans. Ten JC virus-containing founder mice were produced, of which 5 survived to maturity. Four of them developed adrenal neuroblastomas, which metastasized to several other tissues. JC virus tumor-antigen RNA was detected at high levels in the tumor tissues and at low levels in the normal tissues of these mice. One of the three BK virus-containing mice was abnormally shaped and died at 2 weeks of age. The other two BK virus-containing mice developed primary hepatocellular carcinomas and renal tumors and died at 8-10 months of age. BK virus tumor-antigen RNA was expressed in tumor tissues of both mice. Since each of the viruses retained the general tissue tropism that it exhibits in humans, these data suggest that transgenic mice harboring human viruses will be useful as animal models for viral-induced diseases.

The early region of human papovavirus JC induces dysmyelination in transgenic mice.

Transgenic mice containing the early region of human papovavirus JC were produced. Some of these mice exhibited a shaking disorder similar to the previously described mutant mice jimpy or quaking. Neuropathological analysis indicated a dysmyelination in the central nervous system, but not the peripheral nervous system. A high level of JCV T-antigen mRNA was present in the brains of the mice exhibiting the myelin disorder. JC virus is associated in humans with a degenerative demyelinating disease: progressive multifocal leukoencephalopathy. The JCV-containing transgenic mice may therefore provide an animal model for studying this disease.

Dysmyelination in transgenic mice containing JC virus early region.

JC virus (JCV) causes the chronic human demyelinating disease progressive multifocal leukoencephalopathy. Because of host range restrictions, experimental models of JCV-induced demyelination have not been available. The restricted tropism of JCV infectivity has recently been overcome by the production of transgenic mice that contain the early region of JCV in all cells. This portion of the DNA encodes JCV T-antigens. These mice display a dysmyelinating phenotype, the severity of which is related to the level of JCV early region expression in brain. With the use of immunocytochemistry and in situ hybridization, we characterized morphologically myelin-specific and JCV gene expression in a severely affected strain of these mice. Our results suggest that expression of JCV T-antigens occurs predominantly in oligodendrocytes and is the primary cause of dysmyelination. Affected oligodendrocytes do not myelinate axons properly. However, they express myelin-specific genes and display some of the morphological phenotypes of early stages of myelination. A decreased ratio between levels of transcriptional and translational products of genes encoding the major structural proteins of central nervous system myelin was apparent. These results suggest that JCV T-antigens arrest the maturation of oligodendrocytes and inhibit the production of myelin. These results also demonstrate that JCV transgenic mice are a good model for investigating mechanisms of JCV-induced demyelinating lesions in progressive multifocal leukoencephalopathy.

Genetic control of drug resistance: assignment of ama-1 to Chinese hamster chromosome 7, confirmation of assignment of genes coding for TK, GALK, and ACP to chromosome 7, and tentative assignment of TPI to chromosome 8.

The gene which specifies a subunit of RNA polymerase II, ama-1, is assigned to chromosome 7 in the Chinese hamster. The assignment of genes coding for TK, GALK, and ACP to chromosome 7 is confirmed, with a provisional regional assignment of TK and GALK to 7q. On the basis of one clone with six subclones, a provisional assignment of TPI to Chinese hamster chromosome 8 is made. With the assignment of tk and ama-1 to chromosome 7 in the CHO cell line Ama1, this chromosome is shown to have two selectable markers.

Molecular analysis of chromosome-mediated gene transfer.

Metaphase chromosomes isolated from a cell line carrying the thymidine kinase (TK) gene of herpes simplex virus type I were used to transform the TK-deficient cell line LMTK- to the TK+ phenotype. Four independent transformants were isolated, all of which expressed virus-specific TK. Each of the four transformant cell lines initially became TK- at a rate of 12% per day. All four transformants possessed multiple copies of the TK gene and in one of the four a rearrangement occurred adjacent to the TK sequences. Stable TK+ derivatives of each line, isolated after prolonged cultivation, retained fewer copies of the TK gene than did their unstable parents. The transferred chromosomal fragment was larger than 17 kilobases in each line.