Terri Grodzicker

Physical mapping of temperature-sensitive mutations of adenoviruses

Temperature-sensitive mutants of the adenovirus 2-simian virus 40 hybrid, Ad2+ ND1‡, were isolated and crossed with temperature-sensitive mutants of adenovirus type 5 (Ad5). Forty-five wild type recombinants were selected and their DNAs were separately digested with five restriction endonucleases, EcoRI, HpaI, BamI, HindIII and SmaI. There are 15 sites cleaved by these enzymes that are unique to one parental DNA or the other. The fragments obtained by digestion of each of the recombinant DNAs were separated by gel electrophoresis and compared with those obtained from the parental genomes. The recombinant DNAs were found to consist of sequences derived both from Ad5 and from Ad2+ND1. Knowing the positions at which the five restriction enzymes cleave the genomes of the parental serotypes, it was possible to decide which regions of each recombinant DNA are composed of Ad5 and which of Ad2+ND1 sequences. In this way the cross-over events that generated the recombinants were mapped and the physical locations of the ts mutations in the parental genomes were determined.

Mutations that allow human Ad2 and Ad5 to express late genes in monkey cells map in the viral gene encoding the 72K DNA binding protein

Abstract Five host-range mutants (Ad2hr400–hr403, Ad5hr404) of human adenovirus serotype 2 and 5 (Ad2 and Ad5) which overcome the block to growth of wild-type adenovirus in monkey cells have been isolated. They form plaques and multiply efficiently in both monkey and human cells. The alteration in each of these mutants allows the full expression of all viral late genes, in marked contrast to the depressed synthesis of many late proteins in monkey cells infected with the parental Ad2 or Ad5. The altered gene encodes a diffusible product, since the mutation acts in trans to enhance the synthesis of wild-type Ad3 late proteins during co-infections of monkey cells with Ad2hr400 and Ad3. Restriction enzyme analysis of the genomes of all the host-range mutants show that none of them contain major alterations. In addition, an earlier report (Klessig and Hassell, 1978) indicated that Ad2hr400 does not contain SV40 sequences, which in some adenovirus-SV40 hybrid viruses allows efficient multiplication in monkey cells. The mutation responsible for the extended host range has been physically mapped by marker rescue experiments using isolated restriction enzyme fragments of the mutants to transfer the new phenotype to wild-type adenovirus. The alteration in each of the five mutants is located in a region (coordinates 62–70.7; coordinates 62–68 for Ad5hr404) which encodes predominantly the 72K DNA binding protein. More detailed mapping using Ad2hr400 fragments places the mutation (coordinates 62.9–65.6) entirely within the 72K gene. The multifunctional nature of the 72K protein and some of its similarities to SV40 T antigen are discussed.

Adenovirus E1A coding sequences that enable ras and pmt oncogenes to transform cultured primary cells.

Plasmids expressing partial adenovirus early region 1A (E1A) coding sequences were tested for activities which facilitate in vitro establishment (immortalization) of primary baby rat kidney cells and which enable the T24 Harvey ras-related oncogene and the polyomavirus middle T antigen (pmt) gene to transform primary baby rat kidney cells. E1A cDNAs expressing the 289- and 243-amino acid proteins expressed both E1A transforming functions. Mutant hrA, which encodes a 140-amino acid protein derived from the amino-terminal domain shared by the 289- and 243-amino acid proteins, enabled ras (but not pmt) to transform and facilitated in vitro establishment to a low, but detectable, extent. These studies suggest that E1A functions which collaborate with ras oncogenes and those which facilitate establishment are linked. Furthermore, E1A transforming functions are not associated with activities of the 289-amino acid E1A proteins required for efficient transcriptional activation of viral early region promoters.

Expression of SV40 T Antigen under Control of Adenovirus Promoters

We have obtained novel adenovirus-SV40 recombinant viruses that express wild-type SV40 large T and small t antigens under the control of different adenovirus promoters. Hybrids were constructed in vitro with SV40 DNA that contains the entire early coding region but lacks the transcriptional promoter. Recombinants were isolated by a strong biological selection for viruses that express SV40 T antigen. Analysis of several recombinant genomes indicates that they contain the SV40 A gene inserted in a variety of positions and orientations in the adenoviral genome. Moreover, the set of hybrid transcripts reveals an unexpected variety of splicing patterns. Some hybrid mRNAs transcribed from the adenovirus late promoter appear to contain the adenovirus tripartite leader sequence. Other hybrid mRNAs were transcribed from adenovirus early promoters. All recombinant mRNAs contain intact SV40 early sequences that have normal splice patterns and produce wild-type T antigens. Biochemical characterization of SV40 T antigens overproduced by the hybrid viruses indicates that they are structurally indistinguishable from wild-type SV40 large T antigen and are functionally equivalent to the D2 protein.

A retrovirus expressing the 12S adenoviral E1A gene product can immortalize epithelial cells from a broad range of rat tissues.

An epithelial cell-transforming virus could be of great use, both in the culture of epithelial cell lines and in the study of carcinogenesis. Since the adenoviral E1A gene has been shown to partially transform some epithelial cells from primary rat cell cultures, we constructed retrovirus vectors containing either the 12S or 13S E1A cDNA sequences to facilitate the transfer of these genes into a variety of primary cell types. The 12S E1A virus induced proliferation and immortalization of epithelial cells in rat kidney, liver, heart, pancreas, and thyroid primary cultures. In the two cases tested, heart and liver cultures, E1A-immortalized cells were nontumorigenic, but could be completely transformed by subsequent introduction of the ras oncogene. To our surprise, the 13S virus had a greatly reduced immortalization potential. We discuss these data in light of the model of Spindler et al. (K. R. Spindler, C. Y. Eng, and A.-J. Berk, J. Virol. 53:742-750, 1985), in which the 12S E1A protein is required for the complete induction of the cellular DNA replication machinery in the quiescent human epithelial cells in which adenoviruses normally replicate.

A mutation which creates a new site for the re-initiation of polypeptide synthesis in the z gene of the lac operon of Escherichia coli☆

Abstract A class of Mel + revertants of a strongly polar nonsense mutant in the z gene of the lac operon containing a new initiation site for protein synthesis has been isolated and characterized. The new initiation site, called π, operates efficiently to restore permease and acetylase activities to a high level. Translation of the distal portion of the z gene also occurs in these revertants. π is located very close to and on the operator-distal side of the chain-terminating triplet. It can revert other terminator mutants to Mel + if it is incorporated into the z gene close to the operatordistal side of the terminator triplet.

Adenovirus recombination: Physical mapping of crossover events

Abstract Recombinants have been isolated from crosses of temperature-sensitive mutants of two adenovirus serotypes whose DNAs differ in their cleavage patterns with restricting endonucleases. Recombinant and parental genomes were dissected with restriction enzymes and the resulting fragments compared. From the results one can align the adenovirus genetic and physical maps.

Fine structure of the gradient of polarity in the z gene of the lac operon of Escherichia coli

Abstract Peaks have been found in the gradient of polarity of the 2 gene of the lac operon of Escherichia coli . The position of these peaks corresponds to the regions previously shown to contain polypeptide reinitiation sequences.

The location of the genes coding for hexon and fiber proteins in adenovirus DNA

A serological analysis has been made of the capsid antigens hexon and fiber from 17 Ad5-Ad2+ND1 recombinants that enables us to determine the phenotype of the recombinants. By correlation of this data with the genetic and physical maps of the adenovirus genome, obtained by recombination and restriction endonuclease analysis, the genes coding for the hexon and fiber have been assigned to specific locations on the adenovirus DNA.

The physical locations of structural genes in adenovirus DNA

Abstract Interserotypic recombinants are formed in crosses between temperature-sensitive ( ts ) mutants of adenovirus type 5 (Ad5) and the nondefective adenovirus 2-SV40 hybrid virus Ad2 + ND1. The positions of Ad2 + ND1 and Ad5 DNA sequences in recombinant genomes can be determined by analysis of their DNA with restriction endonucleases and the sites of the mutation in many ts mutants have been mapped [Grodzicker, T., Williams, J. F., Sharp, P. A., and Sambrook, J. (1974), Cold Spring Harbor Zymp. Quant. Biol. 39 , 439–446; Sambrook, J., Williams, J., Sharp, P. A., and Grodzicker, T. (1975), J. Mol. Biol. 97 , 369–390]. We show here that several Ad5 polypeptides can be distinguished from their Ad2 + ND1 counterparts by SDS-polyacrylamide gel electrophoresis. Analysis of the polypeptides encoded by recombinants of known genetic constitution has allowed us to locate several structural genes on the adenovirus genetic and physical maps and to assign ts mutants to specific genes. Furthermore, we have physically mapped the early Ad5 mutant ts 125 which has a defect in the gene for the adenovirus-specific single-stranded binding protein. In addition, we show that the virus-associated RNAs of Ad2 and Ad5 can be separated electrophoretically, permitting rapid and simple determination of the origin of the DNA surrounding position 0.30 in interserotypic recombinants.