Transformation of rodent cells by a cloned DNA fragment of herpes simplex virus type 2.

Transformation of rodent cells with isolated restriction endonuclease fragments of herpes simplex virus type 2 DNA identified a region of the genome located between map positions 0.58 and 0.62. These sequences were cloned into pBR322, and the recombinant plasmid was used to transform primary rat embryo cells and NIH 3T3 cells. The transformants were selected for their ability to form dense foci on a monolayer or to form colonies in semisolid medium. In contrast to the parental rat or mouse cells, cell lines transformed with the cloned herpes simplex virus type 2 fragment grow to high saturation densities, replicate in medium containing 1% serum, form colonies in dilute methylcellulose, show reduced levels of fibronectin, and are tumorigenic in nude mice and in their syngeneic hosts. Southern blot hybridizations have detected sequences homologous to the viral fragment in high-molecular-weight DNA from the transformed cell lines that are not present in DNA from normal rodents. In all cases, the plasmid DNA was present in less than one copy per cell, and the patterns of viral sequences changed with passage of the cell line in vivo.     

Polyoma virus mutant with normal transforming ability but impaired tumorigenic potential.

Cloned DNA from the P155 mutant of polyoma virus transforms cells in culture as efficiently as wild-type DNA, but has a much lower tumorigenic potential when injected into newborn rats. Like cells transformed by wild-type DNA, cells transformed by the mutant DNA grow in low serum concentrations, form colonies in agar suspension, and grow to high saturation densities compared with untransformed cells. They are, however, much less tumorigenic since they transplant 100- to 2,000-fold less efficiently than cells transformed by wild-type DNA. Substitution of the region between 89.7 and 1.8 map units by the corresponding region of P155 DNA decreased the tumorigenicity of wild-type DNA. When this region was isolated from wild-type DNA and substituted in P155 DNA, the tumorigenicity of the latter increased to values comparable to those of wild-type DNA. This showed that the lesion affecting tumorigenicity occurred between 89.7 and 1.8 map units on the polyoma virus genome. Sequence analysis in this region revealed a 12-base-pair deletion between nucleotides 1,347 and 1,360. This identified P155 as an mlt mutant, i.e., a mutant with a deletion from a region which encodes parts of the large and middle T antigens.     

Transformation of rat fibroblasts by cloned defective polyoma DNA

Defective polyoma DNA molecules isolated from mouse cells infected with high-multiplicity-passaged virus were cloned in pBR322, and the recombinant plasmids were tested for their capacity to transform Fischer rat 3T3 cells in culture. Recombinants carrying an intact proximal portion of the early region, i.e., the region coding for both small and middle T antigens, were able to induce the transformed phenotype. A recombinant plasmid containing a defective polyoma genome with a deletion of about 300 base pairs in the region coding for the C-terminal segment of middle T antigen failed to transform.     

Polyoma viral middle T-antigen is required for transformation.

To determine whether small or middle T-antigen (or both) of polyoma virus is required for transformation, we constructed mutants of recombinant plasmids which bear the viral oncogene and measured the capacity of these mutants to transform rat cells in culture. Insertion and deletion mutations in sequences encoding small and middle T-antigens (79.7, 81.3, and 82.9 map units) rendered the DNA incapable of causing transformation by the focus assay. Similar mutations in sequences that encoded middle but not small T-antigen (89.7, 92.1, and 96.5 map units) generally abolished the transforming activity of the DNA. However, two mutants (pPdl1-4 and PPd12-7) that carried deletions at 92.1 map units retained the capacity to transform cells; pPdl1-4 did so at frequencies equal to those of the parental plasmid, whereas pPdl2-7 transformed at 10% the frequency of its antecedent. From these studies we conclude that small T-antigen alone is insufficient to cause transformation and that middle T-antigen is required for transformation, either in combination with small T-antigen or by itself.     

Cloning of a DNA fragment from the left-hand terminus of the adenovirus type 2 genome and its use in site-directed mutagenesis.

The HpaI E fragment (0-4.5 map units) of adenovirus type 2 (Ad2) DNA was cloned in the plasmid vector pBR322. Excision of the viral insert with PstI and XbaI generated a fragment which comigrated with Ad2 XbaI-E (0-3.8 map units), and this fragment was ligated to the 3.8-100 fragment generated by XbaI cleavage of the DNA of the Ad5 mutant, dl309 (N. Jones and T. Shenk, Cell 17:683-689, 1979). Transfection with the ligation products resulted in the production of progeny virus which was able to replicate on both HeLa and line 293 cells, demonstrating the biological activity of the sequences rescued from the plasmid. Small deletions were introduced around the SmaI site (map position 2.8) within the cloned viral insert, and the altered DNA sequences were reintroduced into progeny virus as described above. The mutant viruses grew well on line 293 cells but plaqued with greatly reduced efficiency on HeLa cells, exhibiting a host range phenotype similar to previously described mutants with lesions located within this region of the genome. When plasmid-derived left-end fragments containing pBR322 DNA sequences to the left of map position 0 were ligated to the 3.8-100 fragment of dl309 DNA, the infectivity of the ligation products was not reduced. However, all progeny viruses examined yielded normal-size restriction enzyme fragments from their left-hand ends, indicating that the bulk of the pBR322 DNA sequences are removed either prior to or as a consequence of the replication of the transfecting DNA molecules.     

Expression of adenovirus E1a and E1b gene products and the Escherichia coli XGPRT gene in KB cells

The recombinant plasmid pSV2-gpt, which contains the Escherichia coli XGPRT gene under the control of a simian virus 40 early promoter, was modified to contain the type 2 adenovirus (Ad2) XhoI-C (0 to 15.5 map units) restriction endonuclease fragment. Plasmid (pLB206) DNA was introduced into human KB cells by Ca2+-mediated DNA transfection, and transformants were selected in medium containing xanthine, aminopterin, and mycophenolic acid, as a consequence of expression of the dominant, selectable XGPRT gene. A series of 13 gpt+ cell lines were isolated and tested for their ability to complement Ad5 deletion mutants in E1a (H5dl312) and E1b (H5dl315). Four classes of gpt+ KB cell lines were identified, including clones constitutively expressing both E1a and E1b, only E1a, or only E1b or not expressing either E1a or E1b. DNA and RNA filter transfer hybridization analysis substantiated the conclusions that those cell lines capable of complementing viral host range mutants contained the appropriate viral DNA sequences and cytoplasmic polyadenylated RNA species. DNA filter transfer hybridization studies also revealed that the transfected vector DNA was stably integrated into chromosomal DNA in the KB transformants and the number of integrated sites ranged from 1 to 3. The gpt+ KB cell line that only expressed E1b gene functions only contained viral E1b gene sequences; those cell lines that expressed neither E1a nor E1b gene function contained only small or no regions of Ad2 DNA. When weaned off the selective medium, transformed KB cell lines stably maintained their inserted DNA in the absence of selective pressure and could easily be adapted to growth in suspension culture.     

Cloned human polyomavirus JC DNA can transform human amnion cells.

The genome of the human polyomavirus JC (Mad-1 strain) was molecularly cloned in Escherichia coli by using the plasmid vector pBR322. Recombinant DNA molecules were constructed with the entire JC genome inserted either at its unique EcoRI site at 0.0 map units or at its unique BamHI site at 0.51 map units. Viral DNA from each of these recombinant plasmids was capable of transforming human amnion cells, and cell lines established from transformed foci were positive for JC tumor antigen as assayed by indirect immunofluorescence.     

Structural and biological analysis of integrated polyoma virus DNA and its adjacent host sequences cloned from transformed rat cells.

EcoRI fragments containing integrated viral and adjacent host sequences were cloned from two polyoma virus-transformed cell lines (7axT and 7axB) which each contain a single insert of polyoma virus DNA. Cloned DNA fragments which contained a complete coding capacity for the polyoma virus middle and small T-antigens were capable of transforming rat cells in vitro. Analysis of the flanking sequences indicated that rat DNA had been reorganized or deleted at the sites of polyoma virus integration, but none of the hallmarks of retroviral integration, such as the duplication of host DNA, were apparent. There was no obvious similarity of DNA sequences in the four virus-host joins. In one case the virus-host junction sequence predicted the virus-host fusion protein which was detected in the transformed cell line. DNA homologous to the flanking sequences of three out of four of the joins was present in single copy in untransformed cells. One copy of the flanking host sequences existed in an unaltered form in the two transformed cell lines, indicating that a haploid copy of the viral transforming sequences is sufficient to maintain transformation. The flanking sequences from one cell line were further used as a probe to isolate a target site (unoccupied site) for polyoma virus integration from uninfected cellular DNA. The restriction map of this DNA was in agreement with that of the flanking sequences, but the sequence of the unoccupied site indicated that viral integration did not involve a simple recombination event between viral and cellular sequences. Instead, sequence rearrangements or alterations occurred immediately adjacent to the viral insert, possibly as a consequence of the integration of viral DNA.     

Plasmidal maintenance of composite DNA derived from polyoma related plasmid, L factor.

Recently, we reported a multicopy mammalian plasmid with a structure related to polyoma. The plasmid, named L factor, was found at a high copy number (5,000 or more per cell) in a subclone derived from mouse L cells. We attempted to utilize L factor as a plasmid vector for mammalian cells. A series of composite DNA consisting of L factor and a foreign (herpes simplex virus tk) were constructed. These DNA could be established as plasmids after transfection to several mouse cell lines, although the copy number of the re-established plasmids was considerably less than that observed for the original subclone. The composite DNA maintained the structure of the original DNA after prolonged culture and the copy number remained constant even with no selective pressure. A composite DNA, with no DNA sequence corresponding to polyoma T antigen, could also be established as a plasmid in a mouse L cell line in which polyoma T antigen is expressed. The potential use of the plasmid is discussed.     

Cloning of the biotin synthetase gene from Bacillus sphaericus and expression in Escherichia coli and Bacilli

Biotin synthetase (BS) catalyses the biotransformation of dethiobiotin (DTB) to biotin. Here we report the cloning, characterization and expression of the gene encoding BS of Bacillus sphaericus. A recombinant plasmid pSB01, containing an 8.2-kb DNA fragment from B. sphaericus, was isolated by phenotypic complementation of an Escherichia coli bioB strain. Nucleotide sequence analysis of this fragment and N-terminal sequence determination of the recombinant protein product revealed that the bioB gene of B. sphaericus consists of a 996-bp open reading frame which is closely associated with at least one other gene. E. coli cells transformed with a bioB expression vector performed efficient bioconversion of DTB to biotin under defined culture conditions. Biotin production from transformed Bacillus subtilis and B. sphaericus recombinant strains was also demonstrated. Comparison of the amino acid sequences of BS from E. coli and B. sphaericus revealed extensive similarity.     

Transformation of human epidermal cells by transfection with plasmid containing simian virus 40 DNA linked to a neomycin gene in a defined medium

A human epidermal cell culture was transformed by transfection with a recombinant plasmid containing simian virus 40 DNA with a deletion at the origin and an antibiotic (neomycin or G418) marker. A calcium phosphate-mediated DNA transfection method was optimized for introducing exogenous DNA into cells maintained in a fully defined medium. The transformed cells were propagated for more than 200 population doublings and did not appear to go through a "crisis" period. The growth characteristics of the transformed cells were similar to those of untransformed cells. Major keratins synthesized in the transformed cells were similar to those found in normal epidermal cells. Transformed cells initially transfected with the recombinant plasmid could be propagated for more than 30 passages. Actively growing cells could then be repeatedly selected from cell populations based upon their neomycin (G418)-resistant phenotype for at least another 30 passages. Simian virus 40 T-antigen and extrachromosomal DNA containing plasmid- and SV40-specific DNA sequences were detected in the transformed cells. Because of their nononcogenic phenotype and defined growth requirements, the transformed cells provide a model for examining structural changes during cell proliferation and differentiation, and for exploring the multistage carcinogenesis of human epithelial cells.     

Cloning of the crystalline cell wall protein gene of Bacillus licheniformis NM 105.

A protein with a tetragonal pattern, defined as RS protein, was found on the wall surface of an alkaline phosphatase secretion-deficient mutant (NM 105) of Bacillus licheniformis 749/C. The protein was present on the wall surface of the exponential-growth-phase cells, but at the stationary growth phase it was overproduced and hypersecreted. This protein was precipitated to homogeneity from the culture fluid by 80% ammonium sulfate saturation and chilled acetone. The molecular mass of the protein was 98 kilodaltons, and it had a single subunit in a sodium dodecyl sulfate gel. Specific anti-RS antibody was generated in rabbits and used to immunolabel the RS protein on the cells at different growth phases. In early-exponential-growth-phase cells, the outside surface of the wall, the cytoplasm, and the inside surface of the cytoplasmic membrane were labeled. In stationary-growth-phase cells, the cytoplasm was poorly labeled, but the labeling on the outside surface of the wall was high. AB. licheniformis NM 105 gene library was made by using the lambda phage EMBL3. The RS protein expression from this gene library was detected by a modified autoradiographic procedure. One of the amplified RS protein-positive plaques (4213-1) containing recombinant DNA was chosen, and the restriction map of this DNA was prepared. The RS protein expressed in Escherichia coli NM 539 infected with 4213-1 recombinant phage had a lower molecular mass than the purified authentic RS protein. The 4.5-kilobase-pair (kbp) SalI-EcoRI fragment of the recombinant DNA was cloned in the shuttle plasmid pMK4 to construct pMK462, which was expressed in B. subtilis MI112 and produced the RS protein identical in molecular mass to the purified authentic RS protein. The RS protein expression was also demonstrated in cryosections of transformed E. coli and B. subtilis cells by immunoelectron microscopy. The 1.2-kbp SalI-HindIII and 1.8-kbp HindIII-HindIII recombinant DNA restriction enzyme fragments, respectively, from the right of the restriction map produced anti-RS antibody cross-reacting proteins. The expression of the 1.2-kbp SalI-HindIII DNA fragment cloned in pUC8 could be induced with isopropyl-beta-D-thiogalactopyranoside. The 1.8-kbp DNA restriction fragment hybridized with both the chromosomal DNA of strain NM 105 and the recombinant phage 4213-1 DNA. The RS gene expression was finally demonstrated in transformed E. coli 539 cells by in situ hybridization of frozen thin sections with the 1.8-kbp HindIII biotin-dATP probe and immunolabeling these with anti-biotin immunoglobulin G and protein A-gold.     

Replication of plasmid chimera pBR-mtB-A containing rat liver mtDNA fragment in Escherichia coli polA cells

Escherichia coli K-12 strains p108 (polA6), p3478 (polA1), and KS55 (polA12, ts) deficient in DNA polymerase I were transformed by recombinant pBR-mtB-A plasmid containing BamHI-A fragment of rat liver mtDNA and pBR322 plasmid. The physical map of the pBR-mtB-A, containing the recognition sites for SalI, EcoRI and HinIII endonucleases, was constructed and the orientation of mtDNA fragment joined to pBR322 plasmid was studied. The phenotypic selection using ampicillin containing medium at permissive and nonpermissive temperature (KS55 strain), or at 37 °C (polA6 and polA1 strains) revealed that only the cells transformed with the hybrid plasmid are able to grow under these conditions. The presence of mtDNA insertions in chimeric DNA molecules of pBR-mtB-A in polA strains was proved by electrophoretic and hybridization analysis. Thus the results obtained demonstrate the replication of the vehicle containing both plasmid replicon and mitochondrial origin in the conditions nonpermissive for the stable reproduction of the plasmid DNA alone.     

The infectivity of adenovirus genomes lacking DNA sequences from their left-hand termini.

Deletions extending various distances into the left-hand terminal DNA sequences of the adenovirus type 2 (Ad2) genome were generated in a plasmid containing a cloned fragment spanning from 0 to 4.9 map units. The altered Ad2 DNA sequences were introduced into viral genomes by ligating a plasmid-derived fragment, which included the sequences extending to 3.8 map units, to the 3.8-100 map unit fragment generated by XbaI cleavage of the DNA of the Ad5 variant, d1309 (N.Jones and T.Shenk, Cell 17 683-689, 1979). The infectivity of the ligation products was studied by transfection of line 293 cells. Genomes lacking 11, 40, or 51 nucleotides from their left-hand termini, or containing an additional 18dG residues linked to this position were infectious, and analysis of the progeny virus genomes demonstrated that the structure of these modified termini had been restored to normal. In contrast, genomes from which the first 160 base pairs (bp), including the entire 102 bp left hand inverted terminal repeat (ITR), had been removed were non-infectious. The results indicate that the ITRs present at the opposite ends of transfecting DNA molecules are able to interact in vivo, and enable the production of viable viruses containing corrected left-hand terminal sequences. Possible mechanisms for this interaction are discussed.     

State of the viral DNA in rat cells transformed by polyoma virus. I. Virus rescue and the presence of nonintergrated viral DNA molecules.

The interaction of polyoma virus with a continuous line of rat cells was studied. Infection of these cells with polyoma did not cause virus multiplication but induced transformation. Transformed cells did not produce infectious virus, but in all clones tested virus was rescuable upon fusion with permissive mouse cells. Transformed rat cells contained, in addition to integrated viral genomes, 20 to 50 copies of nonintegrated viral DNA equivalents per cell (average). "Free" viral DNA molecules were also found in cells transformed by the ts-a and ts-8 polyoma mutants and kept at 33 C. This was not due to a virus carrier state, since the number of nonintegrated viral DNA molecules was found to be unchanged when cells were grown in the presence of antipolyoma serum. Recloning of the transformed cell lines produced subclones, which also contained free viral DNA. Most of these molecules were supercoiled and were found in the muclei of the transformed cells. The nonintegrated viral DNA is infectious. Its specifici infectivity is, however, about 100-fold lower than that of polyoma DNA extracted from productively infected cells, suggesting that these molecules contain a large proportion of defectives.     

费氏中华根瘤菌与耐盐有关的DNA片段的亚克隆和测序

将费氏中华根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ ｆｒｅｄｉｉ）ＫＴ１９与耐盐有关的２３ｋｂ ＤＮＡ片段用ＢａｍＨⅠ酶切成大小不同的长度,分别与质粒ｐＭＬ１２２连接,然后转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）Ｓ１７－１,筛选出３个转化子。以这些转化子为供体,ＲＴ１９的盐敏感突变株ＲＣ３－３为受体,分别进行二亲本杂交,筛选到接合子ＢＲ２,得到４．４ｋｂ与耐盐有关的ＤＮＡ片段。根据其物理图谱,酶     

Cloning of terminal fragments of the avian adenovirus CELO genome and isolation of a recombinant plasmid carrying the viral oncogene

The terminal fragment of avian adenovirus CELO has been cloned in a plasmid vector. The obtained recombinant plasmid pCBE1 carries the terminal BamHI-E fragment of CELO DNA. Transfection of a nonpermissive culture of Rat2 cell line by the plasmid DNA results in formation of transformation focuses. The cloned BamHI-E fragment of CELO DNA is concluded to contain the viral oncogene. Thus, the CELO genome region deriving the BamHI-E fragment is "left".     

Expression of a gene encoding a rabbit sperm membrane protein in mammalian cells.

A general mammalian expression vector designated pSV2-EP was reconstructed by inserting an oligonucleotide fragment into pSV2-dhfr. This vector allowed insertion of cDNAs with EcoRI cohesive ends. The pSV2-EP contains a simian virus 40 (SV40) early promoter, origin for DNA replication, SV40 poly-A site, splicing site, an initiator ATG downstream from the promoter and an EcoRI site for the insertion of cDNA fragment screened from lambda gt11 expression libraries. A recombinant plasmid (pS-VRS-1) was constructed by inserting RSD-1, a cDNA encoding a rabbit sperm tail protein, into the EcoRI site of the pSV2-EP vector. Chinese hamster ovarian (CHO) dhfr-negative cells were cotransformed with pSV2-dhfr and pSVRS-1 by the calcium phosphate method. In selective culture medium without thymidine and hypoxanthine, several cell lines were obtained containing mRNA and DNA that hybridized with RSD-1. One of these transformed cell lines stained intensely with anti-rSMP-B antibodies, demonstrating that the RSD-1 was expressed in the transformed CHO cells.     

Constitutive and inducible expression of cloned human interferon-b1 gene in HeLa cells through an episomal eukaryotic vector

An EcoRI fragment (1.83 Kb) of human DNA containing B1-interferon coding sequences was cloned in the episomal eukaryotic vector pBK-TK. The recombinant plasmid was transfected into thymidine kinase-deficient HeLa cells. Thymidine kinase-positive transformants were obtained and the cells were found to express B-interferon both constitutively and after induction with Newcastle Disease Virus or poly(rI)-poly(rC). The recombinant plasmid was present in stably transformed HeLa cells in an episomal state in hundreds of copies per cell.