George D. Pearson

Deletion of the Saccharomyces cerevisiae TRR1 Gene Encoding Thioredoxin Reductase Inhibits p53-dependent Reporter Gene Expression

The prevalence of p53 gene mutations in many human tumors implies that p53 protein plays an important role in preventing cancers. Central among the activities ascribed to p53 is its ability to stimulate transcription of other genes that inhibit cells from entering S phase with damaged DNA. Human p53 can be studied in yeast where genetic tools can be used to identify proteins that affect its ability to stimulate transcription. Although p53 strongly stimulated reporter gene expression in wild type yeast, it only weakly stimulated reporter gene expression in Δtrr1 yeast that lacked the gene encoding thioredoxin reductase. Furthermore, ectoptic expression of TRR1 in Δtrr1 yeast restored p53-dependent reporter gene activity to high levels. Immunoblot assays established that the Δtrr1 mutation affected the activity and not the level of p53 protein. The results suggest that p53 can form disulfides and that these disulfides must be reduced in order for the protein to function as a transcription factor.

Replicating adenovirus 2 DNA molecules contain terminal protein

Abstract The type 2 adenovirus chromosome is a linear double-stranded DNA molecule with a 55,000-dalton protein, called terminal protein, covalently joined to each 5′ end. Using a quantitative filter-binding assay for the adenovirus DNA-terminal protein complex, we have shown that adenovirus DNA molecules contain a terminal protein in infected cells. Moreover, we have presented evidence that the terminal protein is linked to parental as well as newly synthesized strands in replicating viral DNA. An equation relating the number of growing points per replicating molecule to the fraction of pulse-labeled DNA retained on filters after cleavage with a restriction endonuclease has been derived. We have calculated an average of 1.6 ± 0.2 growing points per molecule using the equation.

Kinetics of adenovirus dna replication II. Initiation of adenovirus DNA replication

Abstract We have developed three methods to estimate the initiation of adenovirus DNA replication: (a) direct measurement during density-shift experiments, (b) calculation using the saturation kinetics of viral DNA accumulation, and (c) calculation using the steady-state kinetics of viral DNA replication. The values for the rate of initiation measured by each method agree quantitatively and confirm the postulate that initiation is the rate-limiting step in adenovirus replication. Newly replicated viral DNA molecules are preferentially initiated. This preference is slight early in infection, but increases markedly over the course of infection. We interpret these results to mean that adenovirus is partitioned into several pools, and at least one pool contains molecules destined for replication. The average rate of initiation in this pool is 0.0042 ± 0.0007 initiation/min/end and is constant throughout infection. The maximum size of the replication pool is 50,000 ± 7000 molecules/cell. Steady-state calculations indicate that displacement synthesis is faster than complementary synthesis. The differential between these rates of synthesis leads to premature displacement of nascent strands in type I/II replicating molecules, a process we call premature displacement synthesis. We propose that this process provides the raw material for generating defective adenovirus DNA molecules.

Identification, cloning, and R-Loop mapping of the polyhedrin gene from the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata

Polyadenylated RNA was isolated from Orgyia pseudotsugata larvae 8-10 days postinfection with the multicapsid nuclear polyhedrosis virus. This RNA was centrifuged through a sucrose gradient and fractions enriched for polyhedrin mRNA were identified by in vitro translation. Complementary DNA made to this RNA hybridized predominantly to a 5-kb fragment of XhoI-digested viral DNA. This fragment was cloned into the plasmid pACYC177 and mapped with restriction endonucleases. A SalI subclone with a 2.5-kb insert derived from the cloned XhoI fragment was found to select by hybridization only polyhedrin mRNA as determined by the size of the in vitro translation product and its precipitation by anti-polyhedrin antibodies. The orientation of the polyhedrin gene and the region of the insert encoding the N terminus of the polyhedrin protein were determined by DNA sequencing. R-Loop mapping indicated polyhedrin mRNA is 980 +/- 75 bases long and contains about 250 nucleotides not represented in the final protein. The polyhedrin gene had no observable intervening sequences.

Effect of Thioredoxin Deletion and p53 Cysteine Replacement on Human p53 Activity in Wild-type and Thioredoxin Reductase Null Yeast

Reporter gene transactivation by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. To investigate the role of thioredoxin in controlling p53 activity, the level of reporter gene transactivation by p53 was determined in yeast lacking the TRX1 and TRX2 genes encoding cytosolic thioredoxin. Surprisingly, p53 activity was unimpaired in yeast lacking thioredoxin. Subsequent analyses showed that thioredoxin deletion suppressed the inhibitory effect of thioredoxin reductase deletion, suggesting that accumulation of oxidized thioredoxin in mutant yeast was necessary for p53 inhibition. Purified human thioredoxin and p53 interacted in vitro (Kd = 0.9 microM thioredoxin). To test the idea that dithio-disulfide exchange reactions between p53 and thioredoxin were responsible for p53 inhibition in mutant yeast, each p53 cysteine was changed to serine, and the effect of the substitution on p53 activity in TRR1 and Deltatrr1 yeast was determined. Substitutions at Zn-coordinating cysteines C176, C238, or C242 resulted in p53 inactivation. Unexpectedly, substitution at cysteine C275 also inactivated p53, which was the first evidence for a non-zinc-coordinating cysteine being essential for p53 function. Cysteine substitutions at six positions (C124, C135, C141, C182, C229, and C277) neither inactivated p53 nor relieved the requirement for thioredoxin reductase. Furthermore, no tested combination of these six cysteine substitutions relieved thioredoxin reductase dependence. The results suggested that p53 dependence on thioredoxin reductase either was indirect, perhaps mediated by an upstream activator of p53, or was due to oxidation of one or more of the four essential cysteines.

Kinetics of adenovirus DNA replication. I. Rate of adenovirus DNA replication

Abstract The rate of adenovirus DNA replication is constant throughout infection. The average rate of replication is 0.046 ± 0.005 fractional lengths/min or 1600 ± 170 nucleotides/min. The time required to synthesize an adenovirus DNA molecule is 21.7 ± 2.4 min.

Symmetrical adenovirus minichromosomes have hairpin replication intermediates

A special class of panhandles (hairpin or foldback structures) arising from the replication of symmetrical adenovirus (Ad) minichromosome dimers and oligomers have been identified by two-dimensional gel electrophoresis. Hairpins provide evidence for replicative intermediates in the pathway for Ad complementary-strand synthesis. Furthermore, larger inverted sequences give Ad minichromosomes a replicative advantage.

In vitro replication directed by a cloned adenovirus origin

A 5.7-kb recombinant plasmid, called XD-7, contains the terminal XbaI-E fragment from the left end of type 2 adenovirus cloned into the EcoRI site of pBR322. An average of 9% +/- 1% of input supercoiled, protein-free XD-7 DNA replicated as rolling circles with single-stranded tails ranging up to unit length and longer in reaction mixtures containing nuclear and cytoplasmic extracts from adenovirus-infected, but not uninfected, HeLa cells. The adenovirus origin was mapped on XD-7 by electron microscopy at the left boundary of the cloned adenovirus segment. Since replication proceeded rightwards, we conclude that the adenovirus l strand was displaced during replication. No origin was located at or near the EcoRI site on pBR322. Reversing the orientation of the adenovirus origin reversed the direction of replication, and deletion of the adenovirus origin abolished replication.

Size determination of Orgyia pseudotsugata cytoplasmic polyhedrosis virus genomic RNA

Molecular-weight estimates were determined for the Orgyia pseudotsugata cytoplasmic polyhedrosis virus (CPV) genome. The molecular-weight estimates were obtained from measurements of glyoxal denatured RNA molecules following electrophoresis in 1% agarose gels and electron micrographic contour lengths. These estimates were compared to the minimal coding capacity derived from the size of translation products of selected genomic segments. The results obtained from these different experimental approaches were in sufficient agreement to estimate the genome size at 20.4 x 106 (29.8 kbp) with a segmental range from 0.61 to 3.53 x 10(6) (0.89-5.19 kbp). Two other members of the Reoviridae family were examined, the Bombyx mori CPV and the human reovirus type 3 (Dearing strain). The molecular-weight estimates for these two viruses were 20.3 and 19.1 x 10(6), respectively. These results suggest that the previously published molecular-weight estimates for these various double-stranded RNA genomes were underestimated.

Mutational mapping of a cloned adenovirus origin.

We have developed a standardized, quantitative assay to study the function of a cloned adenovirus origin. We have shown that the adenovirus origin is located within the first 20 bp of the adenovirus inverted terminal repetition (ITR), a region containing a sequence conserved among human, simian, murine, and avian adenoviruses. Deletions removing or penetrating from either direction into the conserved sequence inactivated the cloned adenovirus origin. A point mutation within the conserved sequence impaired the adenovirus origin, but point mutations outside the conserved sequence had no effect. These results strongly suggest that the conserved sequence within the first 20 bp of the ITR alone constitutes the adenovirus origin (ori) signal.