Human thymidine kinase gene: molecular cloning and nucleotide sequence of a cDNA expressible in mammalian cells.

A cDNA containing the entire coding region of the human thymidine kinase gene has been molecularly cloned. The cDNA is under the control of a simian virus 40 promoter and is expressible in mammalian cells. The complete nucleotide sequence of the human thymidine kinase cDNA has been determined. The cDNA is 1,421 base pairs in length and has a large open reading frame of 702 base pairs capable of specifying a protein with a molecular weight of 25,504. Genomic Southern blotting experiments show that sequences homologous to the human thymidine kinase cDNA are conserved among many vertebrates, including prosimians (lemur), tree shrews, rats, mice, and chickens. Direct comparison of the nucleotide sequences of the human thymidine kinase cDNA and the chicken thymidine kinase gene reveals ca. 70% overall homology. This homology is extended further at the amino acid sequence level, with greater than 74% amino acid residues matched between the human and chicken thymidine kinase proteins.     

Shuttle vectors conferring hygromycin B resistance to E. coli and to mammalian cells. Differential expression of carboxyterminal fusion proteins.

Shuttle vectors expressing resistance to hygromycin B in both E. coli and in mammalian cells were constructed. A combination of the simian virus 40 early promoter upstream of the native bacterial promoter of the neo gene from transposon Tn5 was found to express hygromycin B resistance better in both types of host cells than a combination of the Tn5 promoter followed by the promoter of the Herpes simplex virus thymidine kinase gene. Hygromycin phosphotransferase fusion proteins with extensions at the carboxyterminus were also tested and found to be marginally less effective as selection markers in eukaryotic cells but virtually inactive in E. coli.     

Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency.

We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (approximately 10(4) base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk-ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.     

Isolation of a simian virus 40 T-antigen-positive, transformation-resistant cell line by indirect selection.

In an attempt to identify cellular genes that might be involved in simian virus 40 (SV40) transformation, we have set out to isolate cells which express T antigen but are not transformed. SV40 DNA and the herpes simplex virus thymidine kinase gene were cotransfected into tk- 3T3 fibroblasts. Of 72 colonies screened that were resistant to hypoxanthine-aminopterin-thymidine, 57 were T antigen positive as judged by immunofluorescence. One of these lines, A27, had a normal growth phenotype in monolayer overgrowth and soft agar assays. It contained intact SV40 sequences that could be rescued by fusion to permissive cells. This rescued virus was fully capable of transforming nonpermissive cells to the same extent as did wild-type virus. The A27 cells, however, were not transformable by infection with SV40 or by transfection of SV40 DNA. It is likely that these cells were altered in a cellular function required for the establishment of the transformed state.     

Shuttle vectors conferring hygromycin B resistance toE. coli and to mammalian cells

Shuttle vectors expressing resistance to hygromycin B in bothE. coli and in mammalian cells were constructed. A combination of the simian virus 40 early promoter upstream of the native bacterial promoter of theneo gene from transposon Tn5 was found to express hygromycin B resistance better in both types of host cells than a combination of the Tn5 promoter followed by the promoter of the Herpes simplex virus thymidine kinase gene. Hygromycin phosphotransferase fusion proteins with extensions at the carboxyterminus were also tested and found to be marginally less effective as selection markers in eukaryotic cells but virtually inactive inE. coli.Abbreviations HM hygromycin - hpt hygromycin phosphotransferase gene - neo neomycin (geneticin) phosphotransferase gene - DHFR dihydrofolate reductase     

Thymidine Kinase from Normal, Simian Virus 40-Transformed and Simian Virus 40-Lytically Infected Cells

Simian virus 40 (SV40) infection of human diploid cells failed to cause an enhanced production of thymidine kinase during the first 10 days after infection. Thymidine kinase activities from extracts of SV40-transformed cultures (human or simian) were considerably higher than the activity levels in extracts from the normal cells of origin. In addition, whereas the kinase activities obtained for human diploid cultures decreased as the cell sheet became confluent, the kinase activities for SV40-transformed human cells remained high after confluence was reached. Antisera obtained from hamsters bearing SV40 or adeno-7-SV40 hybrid virus tumors selectively inhibited enzyme from transformed sources (human or simian). Also, the antisera selectively inhibited enzyme extracted from SV40-lytically infected monkey cells. Sera from normal animals or from hamsters bearing polyoma tumors failed to inhibit enzymes from normal, SV40-transformed, or SV40-lytically infected cells. The Michaelis constant of partially purified enzyme from SV40-transformed cells was two to five times as high as that obtained for partially purified enzyme from human diploid cell cultures.     

DNA-mediated gene transfer in Friend leukemia cells by cotransfection of simian virus 40 DNA with herpes simplex virus thymidine kinase DNA.

Thymidine kinase-negative Friend leukemia cells were cotransfected with simian virus 40 (SV40) DNA and thymidine kinase gene DNA of herpes simplex virus type 1. The transfected thymidine kinase-positive cells were selected in HAT medium, and SV40 T-antigen expression was observed over many months in cells cultured under selective conditions, and after adaptation to normal growth medium under nonselective conditions. It was shown by Southern blot hybridization that SV40 DNA was integrated in multiple copies in the chromosomal DNA of several clones. All SV40 DNA-containing Friend leukemia cell clones analyzed were able to undergo induced erythroid differentiation. Induced cultures still expressed SV40 T-antigen to the same extent that untreated control cultures did.     

Expression of recombinant genes containing herpes simplex virus delayed-early and immediate-early regulatory regions and trans activation by herpesvirus infection.

The promoter-regulatory regions from the herpes simplex virus type 1 (HSV-1) gene for the immediate-early, 175,000-molecular-weight (175K) protein and the HSV-2 delayed-early gene for a 38K protein were linked to the readily assayable bacterial gene for the enzyme chloramphenicol acetyltransferase (CAT). Unexpectedly, in measurements of the constitutive expression of the recombinant genes 40 to 50 h after transfection of Vero cells, enzyme levels expressed from the delayed-early 38K-promoter-CAT construct (p38KCAT) were at least as high as those from the immediate-early 175K-promoter-CAT construct (p175KCAT). In contrast, enzyme levels expressed after transfection of a similar recombinant gene containing a second delayed-early promoter region, that of the HSV-1 thymidine kinase gene, were ca. 20-fold lower. The amounts of enzyme expressed from both p38KCAT and p175KCAT could be increased by up to 20- to 40-fold after infection of the transfected cells with HSV. In comparison, virus infection had no significant effect on enzyme levels expressed from recombinant CAT genes containing the simian virus 40 early promoter region, with or without the 72-base-pair enhancer element. Experiments with the temperature-sensitive mutants HSV-1 tsB7 and HSV-1 tsK indicate that induction of expression from p175KCAT was mediated by components of the infecting virus particle, whereas that from p38KCAT required de novo expression of virus immediate-early proteins. In addition, we show that functions required to induce expression from both p175KCAT and p38KCAT could also be provided by infection with pseudorabies virus and cytomegalovirus.     

Resistance to human immunodeficiency virus type 1 (HIV-1) infection in human CD4+ lymphocyte-derived cell lines conferred by using retroviral vectors expressing an HIV-1 RNA-specific ribozyme.

Toward gene therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections in AIDS, Moloney murine leukemia virus-derived retroviral vectors were engineered to allow constitutive and tat-inducible expression of an HIV-1 5' leader sequence-specific ribozyme (Rz1). These vectors were used to infect the human CD4+ lymphocyte-derived MT4 cell line. The stable MT4 transformants expressing an HIV-1 RNA-specific ribozyme, under the control of the herpes simplex virus thymidine kinase (tk) promoter, were found to be somewhat resistant to HIV-1 infection as virus production was delayed. In cells allowing ribozyme expression under control of the simian virus 40 or cytomegalovirus promoter, the rate of HIV-1 multiplication was slightly decreased, and virus production was delayed by about 14 days. The highest level of resistance to HIV-1 infection was observed in MT4 cells transformed with a vector containing a fusion tk-TAR (trans activation-responsive) promoter to allow ribozyme expression in a constitutive and tat-inducible manner; no HIV-1 production was observed 22 days after infection of these cells. These results indicate that retroviral vectors expressing HIV-1 RNA-specific ribozymes can be used to confer resistance to HIV-1 infection.     

Vectors for efficient expression in mammalian fibroblastoid, myeloid and lymphoid cells via transfection or infection

We have constructed two related types of multi-cloning mammalian expression vectors. The first, pMPSVEH/HE, carries the promoter of the myeloproliferative sarcoma virus (MPSV). This promoter was found to be stronger than both the SV40 early and the trans-activated human immunodeficiency virus promoters in many cell lines including human and rodent fibroblastoid, lymphoid or myeloid cells. The other, pBEH/HE, carries the simian virus 40 (SV40) early promoter and origin of replication. This offers the possibility of encapsidation in SV40 pseudovirions and subsequent gene transfer into, e.g., hemopoietic cells, via infection. The usefulness of the expression systems was tested with a number of genes and cell lines.