Terri Grodzicker and her guidance through the elegance of adenovirus genetics

It is hard to imagine Genes & Development without Terri Grodzicker at the helm. Her steady leadership over several decades established the journal as a leading publication for molecular genetics. Like many others, we have received our fair share of reviews from G&D submissions, with a few papers sneaking into final published form. Through all of this, Terri's hand was apparent—identifying the key message of the paper, holding it up to the standard of the journal, and guiding us to shape the paper in the best way for publication. Our appreciation of Terri, however, also extends to another valuable role: as a mentor back in the early 1980s, well before the advent of G&D. We see her together with us as graduate students, hunched over a table with Terri holding a cup of coffee in one hand and a cigarette in the other. Through clouds of smoke, we are reviewing a summary of the transfections that are under way or puzzling over the restriction maps of recombinant viruses that emerge from our plaque purifications. The long incubations required for transfections and viral growth provided us with many hours like this—times that we remember fondly for the opportunities they provided for us to be mentored by Terri and for the foundation they established for our future careers in science.

Our opportunity to work with Terri arose from the experimental approach underlying our PhD thesis research. We were fortunate to be among the first graduate students in Robert Tjian's lab after he started as an Assistant Professor in the Department of Biochemistry at the University of California at Berkeley in 1979. Together with a group of fellow graduate students and postdocs, the newly established Tij lab was studying the mechanisms of eukaryotic gene regulation with a focus on the SV40 early region, which encodes the SV40 tumor antigen (T antigen). The goal of our work was to test Tij's hypothesis from his postdoctoral studies at Cold Spring Harbor Laboratory that SV40 T antigen negatively regulates its own transcription by binding to three adjacent sites in the early region promoter. The sense of excitement was high because, if successful, this work would provide some of our first insights into the molecular mechanisms of eukaryotic gene regulation. Accordingly, most studies in the lab were focused on DNA binding assays, in vitro transcription, and detailed mutagenesis of the SV40 early promoter (see the group photo below).

The Tjian lab, circa 1980, celebrating the demonstration of autoregulation of SV40 early region transcription (Rio et al. 1980). (Back row from left to right) Alan Robbins (technician), Don Rio (graduate student), Rick Myers (graduate student), Robin Clark (graduate student), Michael Kligman (undergraduate), and Terri Burgess (undergraduate). (Front row from left to right) Carl Thummel (graduate student) and Tij.

In contrast, our thesis projects were designed to establish adenovirus as a system for expressing foreign proteins of interest, with the initial goal of providing an abundant source of SV40 T antigen. At first glance, this project appeared less exciting. Who wants to make the protein that others in the lab would use for their groundbreaking functional studies of DNA binding and gene regulation? As is often the case, however, it is only in retrospect that one realizes the unique opportunities that a project can provide. From a personal perspective, this project established a foundation for true love, which is more than one can expect from most graduate research. We met in the lab, got married a few years later, and have been together ever since. In addition, there was a unique training aspect to this project, since its breadth of experimental work required collaboration outside the lab, leading to our work with Terri.

The best way to achieve our goal was to work with adenovirus–SV40 hybrid viruses, since they can produce massive amounts of T antigen-related protein. This trick relies on the host range of adenovirus, which normally grows in human cells but can be expanded to grow in monkey cells if at least part of SV40 T antigen is expressed (Grodzicker 1980). Indeed, it was a T antigen-related protein called D2, purified from cells infected with the Ad2⁺D2 hybrid virus, that Tij used for his DNA binding studies at CSHL (Tjian 1978). Adenovirus–SV40 hybrid viruses such as Ad2⁺D2 were isolated from mixed populations of adenoviruses and SV40 that had been passaged extensively through monkey cells (Hassell et al. 1978; Grodzicker 1980). The advent of recombinant DNA technology, however, allowed us to move beyond these random viral recombinants to precisely engineer hybrid viruses. This was achieved by molecular cloning followed by the integration of those constructs into an adenovirus vector.

At the time, no one on the Berkeley campus could match Tij's skill in recombinant DNA technology, providing us with a terrific opportunity to design and build recombinant plasmids in bacteria. What we lacked, however, was adequate expertise in adenovirus genetics. Luckily for us, Tij was good friends with Terri from his time at CSHL, so it was only natural that he would turn to her for collaboration on this project. Indeed, there were few others who understood adenovirus genetics to the extent that Terri did, with her many publications on this topic (e.g. Grodzicker et al. 1974, 1975; Sambrook et al. 1975; Williams et al. 1975). Fortunately for us, Terri took this to heart and traveled to Berkeley for weeks at a time to help with our work (see https://www.youtube.com/watch?v=nBItl7sZ0RE for a perspective from Terri on our collaboration).

Our basic approach involved inserting the full-length SV40 T antigen-coding region adjacent to specific segments of adenovirus DNA in a bacterial plasmid. By ligating these constructs to the arms of the adenovirus genome and selecting for growth on monkey cells, we could exploit the high rate of homologous recombination between viral DNAs to precisely target the SV40 DNA within the adenovirus sequences (see diagram above). The initial goal of these efforts, all done in close consultation with Terri, was to provide an abundant source of wild-type T antigen expressed from the strong adenovirus major late promoter (Thummel et al. 1981, 1982, 1983). In addition, these studies provided an unexpected opportunity to characterize the regulation of adenovirus late gene transcription and translation (Thummel et al. 1983; Mansour et al. 1986). Moreover, by simply adding another coding region, we could piggyback on the selection for T antigen to coexpress any foreign protein of interest (Mansour et al. 1985). The ability to grow virus in liter quantities of HeLa spinner cultures combined with the suppression of host cell translation during infection allowed us to produce massive amounts of virally encoded proteins. Indeed, modifications of this concept provide an effective means of producing protein to the present day, most recently exemplified by the use of adenoviruses to develop vaccines against COVID-19 (https://www.youtube.com/watch?v=nBItl7sZ0RE; Jacob-Dolan and Barouch 2022).

Strategy for constructing adenovirus-SV40 hybrids in collaboration with Terri. (Top left) The full-length SV40 T antigen-coding region (SV40) was inserted next to a specific segment of adenovirus DNA (Ad) in a bacterial plasmid. (Top right) This insert was ligated to adenovirus DNA digested with the BamHI restriction enzyme. (Bottom) In this example, a resulting precursor ligation product containing a tandem duplication of adenovirus DNA from regions 11.6 to 17 would undergo homologous recombination to delete the intervening adenoviral DNA, resulting in the final recombinant virus. The placement of the SV40 T antigen-coding region in the adenovirus genome is determined by the adenovirus DNA used in the original bacterial plasmid. This figure is reprinted from Figure 1 in Thummel et al. (1982).

Our work with Terri involved many hours together in the lab, planning experiments, transfecting cells, passaging virus, and mapping recombinant viruses. This last task was perhaps the most fun. Passaging recombinant viruses through monkey cells establishes a strong selection for SV40 T antigen expression. How the virus achieves this goal, however, varies from one genome to the next. Terri guided us through the logic of using Southern blots and restriction sites to solve these recombinant riddles and determine the integrity and position of integrated SV40 sequences (see handwritten note above). Terri's patient explanation of these steps over cups of coffee was an experience we will never forget. These mentoring opportunities also extended to others in the group who spent many hours with Terri going over their latest results, discussing papers, or just sharing science gossip. Many of us formed friendships with Terri that last to the present day.

Terri's notes on plaque-purified adenovirus–SV40 recombinants. This figure lists a number of plaque-purified recombinant viruses that were being mapped by restriction enzyme digestion and Southern blot hybridization.

The advantages of having two mentors for PhD research was particularly evident when it came to writing and presenting our research at meetings. Tij was always the main guide in these efforts. Our presentations would get hammered through rounds of practice talks, ending with trips to the darkroom to develop new slides for the next round (often at night before flying out of town). This could get complicated for the annual tumor virus meeting at CSHL, since Terri would have her own ideas about how the talk could be further improved (thankfully without making new slides). Needless to say, these were experiences that are not easily forgotten but from which we benefited immensely in terms of both the quality of our talks and our approach to mentoring future trainees.

Terri's hand at editing our papers was also, of course, invaluable. A draft of a Results section would be rapidly and repeatedly corrected (without the benefit of a word processing program), and then the hard parts, the Introduction and Discussion, were outlined, discussed over lunch if Terri was in town, or sent back and forth to CSHL for rounds of editing. As the years went by and we saw Terri move on to her leadership role at G&D it all seemed to make sense. Her thoughtfulness, deep understanding of biology, and clarity in thinking and writing have been evident in each issue of the journal. We have all benefited from Terri's remarkable combination of talents and her dedication to scientific publication. G&D could not be in a stronger position as it moves on to new leadership and its next phase as a leading journal for molecular genetics research.