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Y Gluzman

Publications and source records attributed to Y Gluzman.

At least 55 records · Page 3Linked to original sources
PubMed1986

Construction of a helper-free recombinant adenovirus that expresses polyomavirus large T antigen.

Adenovirus-polyomavirus recombinant viruses were constructed in vitro by inserting a hybrid transcription unit composed of the adenovirus type 2 major late promoter and the early coding region of polyomavirus into the adenovirus type 5 vector Ad5 delta E1/dl309. The vector lacks the E1a and E1b transcription units and contains a unique restriction endonuclease cleavage site in their place. The polyomavirus genomic insert contained a small deletion which precluded the synthesis of functional small and middle T antigen but allowed for the synthesis of large T antigen. One recombinant virus, Ad5PyR39, which contained the hybrid transcription unit in the opposite transcriptional orientation from the overall direction of late-gene transcription, was studied in detail. Ad5PyR39 replicated efficiently without a helper virus in human 293 cells and expressed hybrid mRNAs of the expected size and composition that were translated to yield large T antigen. The large T antigen synthesized in 293 cells was the same size as that produced in mouse 3T6 cells lytically infected with polyomavirus, and this protein bound efficiently and specifically to the large-T-antigen-binding sites in polyomavirus DNA. Moreover, the large T antigen encoded by the recombinant virus proved capable of catalyzing the replication in mouse 3T6 cells of a plasmid containing the polyomavirus origin for DNA replication. Comparison of the amount of large T antigen produced in 3T6 cells infected with polyomavirus with that in 293 cells infected with Ad5PyR39, under optimal conditions for each system, revealed at least a fivefold greater yield of the protein on a per cell basis in the latter system compared with the former. Ad5PyR39 should prove to be useful to isolate large quantities of functional polyomavirus large T antigen for structural and biochemical studies.

Adenoviridae↗
PubMed1985

T antigen and template requirements for SV40 DNA replication in vitro.

A cell-free system for replication of SV40 DNA was used to assess the effect of mutations altering either the SV40 origin of DNA replication or the virus-encoded large tumor (T) antigen. Plasmid DNAs containing various portions of the SV40 genome that surround the origin of DNA replication support efficient DNA synthesis in vitro and in vivo. Deletion of DNA sequences adjacent to the binding sites for T antigen either reduce or prevent DNA synthesis. This analysis shows that sequences that had been previously defined by studies in vivo to constitute the minimal core origin sequences are also necessary for DNA synthesis in vitro. Five mutant T antigens containing amino acid substitutions that affect SV40 replication have been purified and their in vitro properties compared with the purified wild-type protein. One protein is completely defective in the ATPase activity of T antigen, but still binds to the origin sequences. Three altered proteins are defective in their ability to bind to origin DNA, but retain ATPase activity. Finally, one of the altered T antigens binds to origin sequences and contains ATPase activity and thus appears like wild-type for these functions. All five proteins fail to support SV40 DNA replication in vitro. Interestingly, in mixing experiments, all five proteins efficiently compete with the wild-type protein and reduce the amount of DNA replication. These data suggest that an additional function of T antigen other than origin binding or ATPase activity, is required for initiation of DNA replication.

Antigens, Polyomavirus Transforming↗
PubMed1985

Transfer of genes into hematopoietic cells using recombinant DNA viruses.

The ability of recombinant DNA viruses to transfer genes into hematopoietic cells has been explored. A recombinant simian virus 40 (SV40) in which the early region had been replaced with the chloramphenicol acetyltransferase (CAT) gene driven by the promoter from Rous sarcoma virus (RSV), was constructed. This virus transferred the CAT gene more efficiently into mouse and human bone marrow cells and into the K562, MEL, and WEHI hematopoietic tissue culture cell lines, than the classical calcium phosphate DNA transfer procedure, as shown by assay for CAT activity 48 hr after infection. Recombinant SV40 virions were also shown to be capable of stably transforming Chinese hamster ovary cells by use of an early region recombinant containing the methotrexate-resistant dihydrofolate reductase (DHFR) gene driven by the RSV promoter. The entire DHFR transcriptional unit could be detected in the genome of transformed cells that were also shown to be resistant to methotrexate. A recombinant adenovirus stock containing the neomycin-resistance gene driven by the SV40 early promoter was used to infect the K562 and MEL hematopoietic cell lines to resistance to the antibiotic G418. Transformation frequency was 10- to 100-fold higher than that obtained with calcium phosphate-precipitated DNA. Most or all of the recombinant adenovirus genome was integrated as 1-3 copies in the transformed cells. These studies show the feasibility of using DNA viruses for introduction of new genetic material into hematopoietic cells.

Acetyltransferases↗
PubMed1985

Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants.

To study the role of the biochemical and physiological activities of simian virus 40 (SV40) large T antigen in the lytic and transformation processes, we have analyzed DNA replication-defective, transformation-competent T-antigen mutants. Here we describe two such mutants, C8/SV40 and T22/SV40, and also summarize the properties of all of the mutants in this collection. C8/SV40 and T22/SV40 were isolated from C8 and T22 cells (simian cell lines transformed with UV-irradiated SV40). Early regions encoding the defective T antigens were cloned into a plasmid vector to generate pC8 and pT22. The mutations responsible for the defects in viral DNA replication were localized by marker rescue, and subsequent DNA sequencing revealed missense and one nonsense mutation. The T22 mutation predicts a change of histidine to glutamine at residue 203. C8 has two mutations, one predicts lysine224 to glutamamic acid and the other changes the codon for glutamic acid660 to a stop codon; therefore, C8 T antigen lacks the 49 carboxy-terminal amino acids. pC8A and pC8B were constructed to contain the C8 mutations separately. Plasmids pT22, pC8, pC8A, and pC8B were able to transform primary rodent cell cultures. T22 T antigen is defective in binding to the SV40 origin. C8B (49-amino-acid truncation) is a host-range mutant defective in a late function in CV-1 but not BSC cells. Analysis of T antigens in mutant SV40-transformed mouse cells suggests that the replicative function of T antigen is important in generating SV40 DNA rearrangements that allow the expression of "100K" variant T antigens in the transformants.

Animals↗
PubMed1985

New host cell system for regulated simian virus 40 DNA replication.

Transformed monkey cell lines (CMT and BMT) that inducible express simian virus 40 (SV40) T antigen from the metallothionein promoter have been isolated and characterized. Immunoprecipitation of pulse-labeled T antigen demonstrates a 5- to 12-fold increase in the rate of synthesis on addition of heavy-metal inducers to the culture medium. Radioimmunoassay of cell extracts indicates the accumulation of three- to fourfold more total T antigen after 2 days of induction by comparison with uninduced controls. A direct correlation was found between the level of T-antigen synthesis and the extent of SV40 DNA replication in inducible cells. Inducible BMT cells expressing a low basal level of T antigen were efficiently transformed by a vector carrying the neomycin resistance marker and an SV40 origin of replication. These vector sequences were maintained in an episomal form in most G418-resistant cell lines examined and persisted even in the absence of biochemical selection. Extensive rearrangements were observed only if the vector contained bacterial plasmid sequences. Expression of a protein product under the control of the SV40 late promoter in such vectors was increased after heavy-metal-dependent amplification of the template. These results demonstrate the ability of BMT cells to maintain a cloned eucaryotic gene in an amplifiable episomal state.

Animals↗
PubMed1985

Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.

Antigens, Polyomavirus Transforming↗
PubMed1984

Infection of eucaryotic cells by helper-independent recombinant adenoviruses: early region 1 is not obligatory for integration of viral DNA.

Recombinant viral genomes carrying a selectable drug resistance marker have been constructed by insertion of a hybrid gene for neomycin resistance into the helper-independent adenovirus vector, delta E1/X. The hybrid gene consists of sequences coding for the aminoglycoside 3'-phosphotransferase II from Tn5, under the control of the simian virus 40 early promoter, and renders mammalian cells resistant to the neomycin analog, G-418. Most of adenovirus early region 1 is deleted from delta E1/X (nucleotides 455 to 3330), and recombinant viral genomes carry the hybrid gene in its place. The large and small XbaI fragments of delta E1/X were ligated to the hybrid gene, and the mixture was transfected into 293 cells. Single plaques were isolated and subsequently passaged in 293 cells to produce virus stocks. The recombinant viruses efficiently rendered cultured rat (Rat2) and simian (CV1) cells resistant to G-418. Cloned cell lines selected for resistance to G-418 contained viral DNA integrated into the host cell genome, demonstrating that early region 1 is not essential for integration of the viral genome. Southern transfer experiments revealed that (i) the sites of integration in the host genome were not unique; (ii) in general, transformed CV1 cell lines contained single-copy, full-length viral genomes, colinear with the infecting virus; (iii) transformed Rat2 cell lines generally contained one to several copies of full-length viral genomes integrated colinearly with the infecting viral DNA; and (iv) three of these five lines of transformed Rat2 cell lines contained tandemly repeated viral DNA sequences in which the right and left ends of the viral genome were joined to each other.

Adenoviruses, Human↗
PubMed1984

Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome.

A variant of the adenovirus type 5 genome which lacks EcoRI sites has been cloned in a bacterial plasmid after the addition of EcoRI oligonucleotide linkers to its ends. Closed circular forms of the recombinant viral genome were not infectious upon their introduction into permissive eucaryotic cells. The linear genome released by digestion of the 39-kilobase recombinant plasmid (pXAd) with EcoRI produced infectious virus at about 5% of the level of wild-type controls. The viruses which arose were indistinguishable from the parental strain, and the normal termini of the viral genome had been restored. Marker rescue experiments demonstrate that provision of a DNA fragment with a normal viral end improves infectivity. When a small fragment carrying a wild-type left end (the 0 to 2.6% ClaI-B fragment) was ligated to ClaI-linearized pXAd, virus was produced with efficiencies comparable to a similar reconstitution of the two ClaI fragments of the wild-type genome. These viruses stably carry the left-end fragment at both ends, leaving the normal right end embedded in 950 base pairs of DNA. The embedded right origin is inactive. The consensus of the analyses reported here is that a free end is a necessary configuration for the sequences which make up the adenovirus origin of replication.

Adenoviridae↗
PubMed1984

Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.

Animals↗
PubMed1984

Efficient transformation of human fibroblasts by adenovirus-simian virus 40 recombinants.

The origin-defective simian virus 40 (SV40) mutant 6-1 has been useful in transforming human cells (Small et al., Nature [London] 296:671-672, 1982; Nagata et al., Nature [London] 306:597-599, 1983). However, the low efficiency of transformation achieved by DNA transfection is a major drawback of the system. To increase the efficiency of SV40-induced transformation of human fibroblasts, we used recombinant adenovirus-SV40 virions which contain a complete SV40 early region including either a wild-type or defective (6-1) origin of replication. The SV40 DNA was cloned into the adenovirus vector in place of early region 1. Cell lines transformed by viruses containing a functional origin of replication produced free SV40 DNA. These cell lines were subcloned, and some of the subclones lost the ability to produce free viral DNA. Subclones that failed to produce free viral DNA were found to possess a mutated T antigen. Cell lines transformed by viruses containing origin-defective SV40 mutants did not produce any free DNA. Because of the high efficiency of transformation, we suggest that the origin-defective chimeric virus is a convenient system for establishing SV40-transformed cell lines from any human cell type that is susceptible to infection by adenovirus type 5.

Adenoviruses, Human↗
PubMed1983

Palindromic adenovirus type 5-simian virus 40 hybrid.

A family of novel adenovirus type 5-simian virus 40 (Ad5-SV40) recombinants (Ad5++D1) whose genomes consist of symmetrically inverted structures was isolated. Particles of Ad5++D1 could contain one of several recombinant genomes that differed incrementally from one another by a full-length copy of linear SV40 DNA. The members of the Ad5++D1 family appeared to be in genetic equilibrium with one another. In all probability this equilibrium was maintained by homologous recombination, resulting in the loss or gain of one or two unit length copies of the SV40 genome. The genome of the most abundant recombinant from consisted of a giant inverted repeat which was some 35,000 nucleotide pairs in length. Beginning from one end, the recombinant genome consisted of 3,534 nucleotides derived from the left end of the adenovirus type 5 genome; these nucleotides were joined to 2.7 copies of SV40 DNA arranged as head-to-tail tandems. This entire structure was then repeated in the opposite orientation, thereby forming a large inverted repeat whose structure was Ad5-SV40-SV40-04VS-04VS-5dA. The population of hybrid genomes was stable and was maintained through serial rounds of infection.

Adenoviruses, Human↗
PubMed1983

DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication.

Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.

Animals↗
PubMed1982

Sequence of the junction in adenovirus 2-SV40 hybrids: examples of illegitimate recombination.

The nucleotide sequences of six Ad2-SV40 junctions from three Ad2-SV40 hybrid viruses (Ad2++HEY, Ad2++LEY and Ad2+D1) were determined. Comparison of parental adenovirus 2 and SV40 DNA sequences with the sequence at the Ad2-SV40 junctions revealed that 5 out of 6 junctions are abrupt transitions from Ad2 to SV40 DNA, and in one case (Ad2++LEY, right junction) there is an additional nucleotide at the junction, which cannot be ascribed to either DNA. Ad2++HEY and Ad2+D1 right junctions are identical and Ad2++LEY and Ad2+ND4 left junctions are identical, a result that strongly suggests these Ad2-SV40 hybrids arose by recombination between the linear Ad2 DNA and circular SV40 DNA, followed by recombination between Ad2 DNA and SV40 DNA present in the Ad2-SV40 hybrid DNA. The unambiguous transition of Ad2 DNA into SV40 DNA at the junction sites is an example of recombination events which have apparently occurred without any homology at the recombination site.

Adenoviruses, Human↗
PubMed1981

SV40-transformed simian cells support the replication of early SV40 mutants.

CV-1, an established line of simian cells permissive for lytic growth of SV40, were transformed by an origin-defective mutant of SV40 which codes for wild-type T antigen. Three transformed lines (COS-1, -3, -7) were established and found to contain T antigen; retain complete permissiveness for lytic growth of SV40; support the replication of tsA209 virus at 40 degrees C; and support the replication of pure populations of SV40 mutants with deletions in the early region. One of the lines (COS-1) contains a single integrated copy of the complete early region of SV40 DNA. These cells are possible hosts for the propagation of pure populations of recombinant SV40 viruses.

Cell Line↗
PubMed1981

Identification of DNA sequences required for transcription of the human alpha 1-globin gene in a new SV40 host-vector system.

We have developed a rapid and simple method for studying the transcription of cloned eucaryotic genes, which involves transfecting SV40-transformed monkey cell lines (COS cells) with derivatives of the plasmid pBR322 that contain the SV40 viral replication origin but lack regions necessary for viral transcription (SV-ORI vectors). Because COS cells produce SV40 T antigen and are permissive for SV40 viral replication, transfected SV-ORI plasmids replicate to a high copy number. SV-ORI plasmids carrying a human alpha-globin gene are also replicated in COS cells. Moreover, the alpha-globin gene is faithfully transcribed to produce high levels of RNA, which is accurately processed to produce authentic alpha-globin mRNA. We have used this transcription system to demonstrate that a sequence located between 55 and 87 base pairs upstream from the mRNA capping site is required for efficient transcription of the alpha-globin gene in COS cells.

Animals↗
PubMed1981

Identification of a promoter component involved in positioning the 5' termini of simian virus 40 early mRNAs.

The 5' termini of the principal early mRNAs produced in cells transformed by wild-type simian virus 40 lie 21-25 nucleotides downstream from an A-T-T-T-A-T sequence on the DNA template. The 5' termini of early mRNAs produced by five origin-defective mutants containing deletions downstream from the A-T-T-T-A-T sequence and one viable mutant dl892 with a deletion starting 15 nucleotides upstream from this sequence were determined by a method involving synthesis, separation, and determination of the sequences of DNAs complementary to 5' termini. Mutant dl892 produced early mRNAs with the same principal 5' termini as wild-type virus; the origin-defective mutants produced mRNAs with principal 5' termini shifted downstream by a distance equivalent to the length of the deleted DNA segment. These data suggest that a DNA sequence of 29 nucleotides, which includes the A-T-T-T-A-T sequence, contains a component(s) of a promoter for early transcription. This component functions in positioning the 5' ends of the principal early mRNAs 21-25 nucleotides downstream from the A-T-T-T-A-T sequence and acts independently of these downstream sequences.

Base Sequence↗