Biomedical subjects

F L Graham

Publications and source records attributed to F L Graham.

At least 145 records · Page 8Linked to original sources

PubMed1989

Insertional mutagenesis using a synthetic lac operator.

We have developed a novel cassette for generating insertion mutants in multi-copy bacterial plasmids. The cassette consists of synthetic oligodeoxyribonucleotides (oligos) which form a DNA duplex following reconstitution in vitro, due to sequence complementarity. It contains a 21-bp segment of the lac operator (lacZo), to provide a readily detectable phenotypic marker. Bacterial colonies harboring plasmids with insertions of this cassette are blue due to constitutive expression of the lac operon resulting from titration of lac repressor molecules by plasmid-borne lacZo sequences. Synthetic oligos containing a desire sequence may be added to the cassette by complementary ends for targeted insertion into plasmids. Sequencing of the resulting insertion mutants is facilitated by using oligos within the cassette as primers for bidirectional sequencing. This allows a complete characterization of each insertion in terms of location, structure of flanking sequences, and orientation of the inserted oligo. We have used this system to construct a series of mutants in early region 1a genes of human adenovirus type 5. For this purpose we designed a cassette which had all three possible translational reading frames open when inserted in one orientation, and all reading frames closed in the other orientation. The cassette also had BamHI restriction sites at each end which could be used to 'collapse' mutants, reducing the size of each insert to 6 bp.

Base Sequence↗

PubMed1989

Infectious circular DNA of human adenovirus type 5: regeneration of viral DNA termini from molecules lacking terminal sequences.

A series of plasmids containing the entire human adenovirus genome with viral DNA termini joined 'head to tail' has been isolated. Several plasmids were able to generate infectious virus following transfection of human cells in spite of having small deletions and rearrangements at the junctions of termini. One plasmid has lost 2 bp of DNA from one end of the viral genome and 11 bp from the other end yet produced viruses with complete wild-type sequences at both ends of the genome. We propose a model for replication of viral DNA off circular templates in which regeneration of terminal information involves translocation of primer and polymerase during initiation of DNA replication. The model suggests a novel mechanism for extension of the 5' ends of linear DNA molecules which could be applicable to chromosomal telomeres.

Adenoviruses, Human↗

PubMed1989

Protection of mice against lethal challenge with herpes simplex virus by vaccination with an adenovirus vector expressing HSV glycoprotein B.

Increasing attention has been focused on the use of recombinant mammalian viruses as potential vaccines. Recombinant human adenoviruses are one of the more promising vaccine vectors because they can be easily constructed and because live adenovirus vaccines have been administered orally to large numbers of military recruits without adverse reactions. In order to examine the efficacy of human adenoviruses as vaccines we have studied the immunity induced by a recombinant adenovirus vector, AdgB2, which induces high level expression of herpes simplex virus (HSV) glycoprotein B (gB) in human and murine cells. Mice inoculated with AdgB2 produced antibodies specific for gB which neutralized HSV in the presence of complement. Although mice inoculated with AdgB2 showed no ill-effects after AdgB2 inoculation and we were unable to detect replication of human adenoviruses in mice, the mice were protected from a lethal challenge with HSV after a single inoculation with AdgB2.

Adenoviruses, Human↗

PubMed1989

The effect of E1 mutations on biochemical transformation by an adenovirus carrying the herpes simplex virus thymidine kinase gene in region E3.

A series of human adenovirus type 5 (Ad5) vectors has been constructed in which a vector containing the human herpes simplex virus thymidine kinase (TK) gene has been recombined with several Ad5 early region 1 (E1) mutants. The resulting viruses were used to study host-virus interactions in TK- rat cells and to examine the importance of E1 functions in a biochemical transformation assay. One of the most important parameters affecting transformation efficiency in this system was the cytotoxicity of the transforming virus. Ad5 viruses expressing the E1a 289 amino acid protein were all highly cytotoxic and induced significantly fewer colonies than did less cytotoxic mutants which were defective in expression of the 289 amino acid product. When correction was made for differential cell viability the variation in transformation efficiencies was considerably reduced although some E1a mutants still demonstrated an enhanced ability to transform in comparison to wt virus. The significance of these results to morphological transformation by adenoviruses is discussed.

Adenoviruses, Human↗

PubMed1989

Expression of the glycoprotein of vesicular stomatitis virus by infectious adenovirus vectors.

We have constructed recombinant human adenovirus (Ad) vectors containing the glycoprotein gene of vesicular stomatitis virus (VSV). The structural gene of the VSV glycoprotein was modified by the addition of promoter and poly(A) addition sequences from the herpes simplex virus type 1 thymidine kinase (TK) gene and inserted, in either orientation, into early region 3 (E3) of human Ad type 5. The recombinant vectors were fully infectious and replicated in HeLa cells in culture. The TK promoter was functional in both insert orientations and responsive to trans-activation by herpes virus infection; however production of VSV glycoprotein in readily detectable amounts was only obtained with the vector having an insert in the E3 parallel orientation (AdG12), and depended principally on transcripts initiating within upstream Ad sequences. The onset of expression of the glycoprotein in AdG12-infected cells was detectable at about the same time as the Ad 72K DNA-binding protein encoded by E2, and its synthesis was not prevented by blocking viral DNA synthesis. The VSV glycoprotein produced by AdG12 was fully processed and could function to direct low pH-induced fusion of infected cells. These Ad vectors have considerable potential utility for the expression of antigens in cell culture and for the immunization of animals in studies of immunity and protection.

Adenoviruses, Human↗

PubMed1989

Use of human adenovirus-based vectors for antigen expression in animals.

An infectious recombinant human adenovirus type 5 (Ad5) vector, AdG12, which carries the glycoprotein gene of vesicular stomatitis virus (VSV) and expresses that gene in cultured HeLa cells was used to examine the host range of insert expression by human Ad vectors. The VSV glycoprotein was expressed in bovine, canine and murine cells when infected with AdG12 in culture. These cell lines are respectively permissive, non-permissive and semi-permissive for human Ad5 replication. Administration of the AdG12 vector to calves, piglets or dogs by either the subcutaneous or oral route resulted in the production of high titres of neutralizing antibodies to VSV. Mice injected intraperitoneally with the vector produced neutralizing antibodies and were protected against subsequent intravenous challenge with normally lethal doses of VSV. This work demonstrates the utility of human adenoviral vectors for antigen expression in a number of non-human cell lines and for the induction of an immune response to the delivered antigen in a number of species.

Adenoviruses, Human↗

PubMed1989

Adenoviruses with nonidentical terminal sequences are viable.

Adenovirus genomes consist of linear DNA molecules containing inverted terminal repeat sequences (ITRs) of 100 to 200 base pairs. The importance of identical termini for viability of adenoviruses was investigated. The viral strains used in this study were wild-type adenovirus type 5 (Ad5) and a variant Ad2 strain with termini which were distinct from those of all other human adenoviruses sequenced to date. A hybrid virus (sub54), obtained by recombination between Ad2 and Ad5, derived the left 42 to 52% of its genome from Ad2 and the right 58 to 48% from Ad5. Southern blotting analysis with labeled oligodeoxynucleotides indicated that both Ad2 and Ad5 ITRs were present in sub54 viral DNA preparations, and successive plaque purifications of sub54 demonstrated that viruses with nonidentical terminal sequences were viable but were rapidly converted to viruses with identical ends. Cloning of the sub54 genome as a bacterial plasmid supported the observations made by analysis of sub54 virion DNA. A plasmid, pFG154, was isolated which contained the entire adenovirus genome with an Ad2 ITR at the left terminus covalently linked to an Ad5 ITR at the right terminus. Upon transfection of mammalian cells with pFG154, viral progeny were obtained which had all possible combinations of termini, thus confirming that molecules with nonidentical termini are viable. Pure populations of viruses with nonidentical termini could not be isolated, suggesting efficient repair of one end with the opposite terminus used as a template. A model for this process is proposed involving strand displacement replication and emphasizing the importance of panhandle formation (annealing of terminal sequences) as a replicative intermediate.

Adenoviruses, Human↗

PubMed1988

A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5.

Early region 1 (E1) of the human adenoviruses has many intriguing properties which have prompted numerous mutational studies to help delineate and characterize the domains responsible for these functions. In mutational analyses being done currently, the E1 region is usually cloned into a bacterial plasmid where it is mutated and then the altered E1 sequences are "rescued" back into infectious virus. The most frequently used rescue procedures are somewhat tedious, requiring the purification and fractionation of linear viral DNA or DNA fragments, and often involve the screening of numerous plaque isolates. Several observations we have made recently on the properties of adenovirus DNA in infected cells and on infectious plasmids in transfected cells led us to design a new approach for rescuing E1 mutations into infectious viral genomes. We constructed a plasmid, pJM17, containing the entire Ad5 DNA molecule, with an insert in the E1 region that exceeds the packaging constraints of the adenovirus capsid. Following transfection of pJM17 into 293 cells the plasmid DNA is able to replicate but cannot be packaged into infectious virions. In contrast cotransfection of 293 cells with pJM17 plus an E1-containing plasmid carrying mutated sequences produces recombinant virions at high efficiencies. Neither plasmid needs to be linearized prior to contransfection. The technique eliminates the need to purify and manipulate infectious virion DNA and since no unique restriction sites are needed, both E1A and E1B mutants' as well as foreign gene inserts in the E1 region can be easily rescued into virus.

Adenovirus Early Proteins↗

PubMed1988

Abundant expression of herpes simplex virus glycoprotein gB using an adenovirus vector.

Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is a major component of infected cell membranes and virion envelopes. Glycoprotein B is known to be essential for entry of viruses into cells and may play important roles in virus-induced cell fusion and other alterations in cell morphology. In order to study the biochemical and immunological properties of gB in isolation from other HSV-1 polypeptides we have constructed human adenovirus vectors capable of expressing high levels of gB. The gB gene was coupled to the SV40 early promoter and inserted into the E3 region of two adenovirus vectors, one in which the E1 region was deleted (AdgB-1) and another which contained E1 sequences (AdgB-2). In AdgB-1 the orientation of the chimeric gB-SV40 gene was right to left, i.e., opposite to the direction of late and E3 mRNA transcription, whereas in AdgB-2 the orientation was left to right. Human 293 cells which express E1 functions supported replication of AdgB-1 and gB was expressed in these cells but not in mouse cells and only at very low levels in human cells other than 293. Replication of AdgB-2 was not limited to 293 cells and the virus was able to induce synthesis of gB at levels equal to or higher than those expressed in HSV-1-infected human or mouse cells. Microscopic examination of AdgB-2-infected cells revealed extensive vacuolization in a manner completely uncharacteristic of adenovirus-infected cells, and fluorescent antibody staining indicated that gB was not only present at the cell surface but also concentrated in the cytoplasmic vacuoles.

Adenoviridae↗

PubMed1987

Stable transfer of a mouse dihydrofolate reductase gene into a deficient cell line using human adenovirus vector.

A plasmid containing the mouse dihydrofolate reductase (dhfr) gene was rescued in a human adenovirus in early region 3. Analysis of the insert in the recombinant virus revealed that the dhfr sequences were intact in the viral genome, whereas a part of the ampicillin gene in the plasmid sequences was deleted. The recombinant virus could successfully express this gene in a deficient cell line. A permanent dhfr+ cell line was established by stable transfer of the gene using the recombinant virus.

Adenoviruses, Human↗

PubMed1987

Protein IX, a minor component of the human adenovirus capsid, is essential for the packaging of full length genomes.

Human adenovirus type 5 (Ad5) contains a 36-kb double-stranded DNA molecule in an icosahedral capsid. Attempts to construct Ad5 insertion mutants containing DNA of more than about 105% of the genome size resulted in viral progeny in which deletions had occurred suggesting the existence of severe constraints on the size of packageable DNA molecules. To partially circumvent these constraints we used an adenovirus vector, Ad5dlE1,3, with deletions in early regions 1 (E1) and 3 for a total net reduction in genome size of 5349 bp and an expected capacity for inserts of greater than 7 kb. To use this vector efficiently we generated a circular form of dlE1,3 DNA which could be propagated as an infectious bacterial plasmid. When this plasmid was used as a recipient for inserts of various sizes it was found that its capacity was much less than expected and that dlE1,3 virion capsids could not even package DNA as large as the wt genome. Because the E1 deletion of dlE1,3 extends into the coding sequences for protein IX, a minor capsid component known to affect the heat stability of adenovirions, the possibility that absence of this polypeptide might also affect the DNA capacity of the virion was investigated. It was found that when the coding sequences for protein IX were restored the packaging capacity of the vector was also restored to that of wt virions. Thus protein IX is an essential constituent of virion capsids dispensable only for virions containing DNA of less than genomic size.

Adenovirus Early Proteins↗

PubMed1987

Growth of 293 cells in suspension culture.

A subline of 293 cells able to grow in suspension culture has been developed by passage of 293 cells through nude mice. This new line, designated 293N3S, grows with a doubling time of approximately 30 h, continues to express adenovirus 5 early region 1 (E1) antigens, and remains permissive for adenovirus 5 host range mutants defective in E1 functions.

Adenoviruses, Human↗

PubMed1986

Characterization of an adenovirus type 5 mutant carrying embedded inverted terminal repeats.

During construction of an adenovirus type 5 (Ad5) deletion mutant, dlE1,3, lacking E1 and E3 sequences, we isolated a variant, dlE1,3-1, which had a direct repeat of viral DNA terminal sequences attached to the left end of the genome. Analysis of this variant with restriction enzymes and by hybridization of Southern blots with specific probes indicated that the extra terminal segment contained the left 2.6% (920 bp) of Ad5 joined to 352 bp of pBR322 which in turn was linked to the left end (minus 21 bp) of dlE1,3. During replication of dlE1,3-1 the extra terminal segment was found to transfer to the right end of the genome resulting in a second variant, dlE1,3-2, with duplicated terminal sequences at both ends of the viral genome. DlE1,3-2 in turn was shown to revert back to dlE1,3-1 at high frequency. Although evidence was obtained indicating that the extra segment could be lost from the left end, spontaneous mutants which had lost direct repeats from both ends were never isolated. It was, however, possible to remove the extra terminal repeat of dlE1,3-1 by cleavage with a restriction enzyme and to isolate dlE1,3 containing wt termini. The rearrangements occurring during replication of dlE1,3-1 and dlE1,3-2 may be consequences of the mode of replication of Ad5 DNA and bear some resemblance to segment inversion in herpesviruses.

Adenoviruses, Human↗

PubMed1986

Human adenovirus cloning vectors based on infectious bacterial plasmids.

By making use of the fact that human adenovirus DNA circularizes in infected cells, and that circular forms of the viral genome are infectious, we have developed an improved adenovirus-based cloning system. A deletion mutant of adenovirus type 5 (Ad5) with deletions in early regions 1 (E1) and 3 (E3) was converted to a bacterial plasmid which can regenerate infectious virus following transfection into human 293 cells. A single XbaI recognition site in the deleted E3 region serves as a site for the insertion of foreign DNA. We have used this system to clone a number of genes into the Ad5 genome and describe the insertion of the neomycin/G418 resistance marker into Ad5 as an example.

Adenoviruses, Human↗

PubMed1986

Expression of the adenovirus E1A oncogene during cell transformation is sufficient to induce susceptibility to lysis by host inflammatory cells.

Mammalian cells transformed by nononcogenic human adenoviruses exhibit high susceptibility to destruction by host mononuclear inflammatory cells. We have analyzed the viral gene regulation of the susceptibility of transformed cells to lysis by natural killer cells and activated macrophages. Comparisons of target cell lines transformed by overlapping segments of the adenovirus E1-transforming gene region revealed that isolated expression of a single oncogene, E1A, was sufficient to cause increased cytolytic susceptibility in the absence of detectable transformed cell-surface expression of viral transplantation antigens and irrespective of histocompatibility antigen identity between killer cells and target cells. These results suggest that oncogene functions that are not linked to the expression of previously recognized cell-surface target structures may actively induce neoplastic cell elimination by components of the host immune surveillance system.

Adenovirus Early Proteins↗

PubMed1986

Development of a helper-independent human adenovirus vector and its use in the transfer of the herpes simplex virus thymidine kinase gene.

Approximately 2 kilobases (kb) of additional DNA can be packaged into wild-type virions of human adenovirus type 5 (Ad5). To extend this limit, a helper independent Ad5 cloning vector was constructed by deleting most of early region 3 (E3) from map coordinates 78.5 to 84.7 and essentially all of early region 1 (E1) from coordinates 1.0 to 10.6. E3 is nonessential for adenovirus replication in cultured cells, and E1 is nonessential when the virus is propagated in 293 cells which constitutively express the E1 gene products. The resulting new virus, dlE1,3 is about 5.5 kb shorter than wild-type Ad5 and therefore should be able to accept up to 7.5 kb in foreign DNA. To test the usefulness of this vector, the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (tk) along with its regulatory sequences was inserted into the unique XbaI site of dlE1,3 (at map position 78.5/84.7). The resulting recombinant virus, Adtk, expressed the HSV tk at a low level (as compared with HSV-1) in infected cells; however, tk expression was markedly enhanced when Adtk-infected cells were superinfected with a tk- mutant of HSV. Furthermore, the Adtk virus efficiently transformed tk- mouse cells (line LTA) to the tk+ phenotype. At a low efficiency, it was also possible to transform tk- human cells (line 143), and tk+ transformants of both mouse and human origin have been established as permanent lines.

Adenoviruses, Human↗

PubMed1985

Transformation by human adenoviruses.

When, approximately 10 years ago, it was shown that the functions essential for cell transformation were localized in a small region of the adenovirus genome, a DNA segment which at that time was thought to be capable of encoding two or three average-sized proteins at most, it seemed reasonable to hope that an understanding of the mechanisms by which adenoviruses transform cells might be quickly achieved. While such optimism might be forgiven, it was quite clearly naive in the extreme. As a consequence of mRNA splicing and the use of overlapping reading frames the number of proteins encoded within E1 is 2-3-times greater than would have been predicted a decade ago, and post-translational modifications may add another dimension of complexity. In fact it has taken nearly all of the past decade just to identify the proteins encoded in E1 and to characterize them in the most rudimentary way. However, we have now entered a period in which new information is accumulating at an extremely rapid rate as a result of several major technical and fundamental advances. Chief among these are the use of recombinant DNA techniques, particularly site-directed mutagenesis, which combined with methods for introducing mutations made in cloned sequences back into infectious virus, clearly represents a powerful approach to studying the functions of transforming proteins. In addition, the ability to express transforming proteins in bacteria and to produce large amounts of highly purified proteins which previously were only just detectable in infected and transformed cells is a major breakthrough. Advances in immunological techniques, particularly the development of monoclonal antibodies and antisera against synthetic peptides, have enormously simplified the task of detecting and characterizing E1 proteins. Finally, recent results suggesting that adenovirus transforming proteins may be functionally and structurally similar to other oncogenes brings a new perspective to the study of oncogenic transformation. Have all the proteins involved in transformation by adenoviruses been identified? It seems probable that all those virally coded proteins which play a major role are now known but of course minor players in the cast could still be waiting in the wings. We have pointed out that viral functions encoded outside region E1 may have some importance at least in initiation of transformation by virions and have speculated on the possibility that one or more of these may be involved in the integration of viral DNA into the host cell chromosome.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoviruses, Human↗

PubMed1985

Nonrandom insertion of Tn5 into cloned human adenovirus DNA.

The bacterial transposable element Tn5 displays regional selectivity in target sites for transposition. To examine this integration specificity of Tn5, we have mapped 57 insertion events in a plasmid pXC1 containing a eukaryotic viral DNA fragment as a target for Tn5 insertional mutagenesis. We found a nonrandom distribution of integration sites in pXC1, suggesting preferred targets for transposition. However, DNA sequence analysis of seven mutants revealed no target site sequence specificity for Tn5 insertion. We demonstrated that the majority of these insertions mapped downstream from a fortuitous promoter sequence which was present and active in this cloned insert in pXC1. Furthermore, when this promoter region was removed, Tn5 was able to transpose into previously unused upstream target sequences. Our data suggest that transcriptional activity may influence Tn5 transposition.

Adenoviruses, Human↗