The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV), which is used for the overexpression of eukaryotic genes and is being engineered for possible use as a viral insecticide, has a circular, supercoiled genome of approximately 128 kilobases. Despite its widespread use, little is known about the mechanism by which AcMNPV replicates. Evidence is presented in this report that AcMNPV origins of DNA replication are repeated sequences each containing several closely related imperfect palindromes that are present in six regions distributed around the genome. Although AcMNPV infection-dependent plasmid replication was initiated by a single complete palindrome, the amount of replication was substantially increased in plasmids containing six or eight palindromes.

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 ⌬trr1 yeast that lacked the gene encoding thioredoxin reductase. Furthermore, ectoptic expression of TRR1 in ⌬trr1 yeast restored p53-dependent reporter gene activity to high levels. Immunoblot assays established that the ⌬trr1 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.Mutations in the p53 tumor suppressor gene have been found in more than half of human cancers. Of 5000 mutations documented, about 85% are missense mutations and greater than 95% are found in the DNA-binding core domain. Ectopic expression of wild type p53 in cells with p53 null mutations suppresses cell growth; expression of oncogenic forms does not (1). Moreover, oncogenic forms of p53 inhibit the ability of wild type p53 to stimulate reporter genes (2). Human tumors frequently express large amounts of p53 antigen. Mice homozygous for p53 null mutations develop normally but show a high tumor incidence (3). These and other results suggest that although p53 null mutations are oncogenic, oncogenicity is most frequently due to dominant negative mutations that result in production of a defective p53 that interferes with the function of wild type p53.It is becoming increasingly clear that cancer results from defects in cell cycle control. Our understanding of cell cycle control derives largely from work first initiated in yeast. All eucaryotic cells have cyclins and cyclin-dependent protein kinases (CDKs) 1 that initiate and orchestrate the transition from one cell cycle compartment to the next. In addition to CDKs, all cells have checkpoint systems that prevent CDKs from initiating the next cell cycle phase until critical processes in the current phase are complete (4). One major checkpoint mechanism is mediated by a set of proteins called cyclin-dependent kinase inhibitors, which bind and inactivate CDKs.In both yeast and mammalian systems, initiation of S phase is delayed when G 1 cells are UV-irradiated. In mammalian cells lacking p53 (or in cells with dominant negative p53), the delay in S phase does not occur, and cells enter S phase with damaged DNA. If wild type p53 is present, the cells enter S phase only when the damage is repaired, or, if the damage is too severe, the cells undergo apoptosis. The discovery that p53 stimulates transcription of the cyclin-dependent kinase inhibitor gene CIP1, which encodes a G 1 cyc...

A gene coding for proteinase inhibitor I, whose expression is induced in tomato leaves (Lycopersicon esculentum L. var. Bonny Best) in response to wounding or insect attacks, was isolated from a genomic library and characterized. The nucleotide sequence revealed that the gene is complete and encodes the sequence of an inhibitor I cDNA that was previously isolated from a cDNA library prepared from wound-induced mRNA from tomato leaves. This gene is located 13.1 kilobase pairs (kbp) upstream from an inhibitor II gene. The wound-inducible gene is interrupted by two intervening sequences of 445 and 404 bp, situated within the codons of amino acids 17 and 47, respectively, of the open reading frame. In addition to the presence of putative regulatory sequences, TATAAA and CCACT, two copies of an imperfect direct repeat approximately 100 bp long were identified in the 5'-flanking region. Phylogenetic comparisons of wound-inducible inhibitor I genes within the genomes of various Lycopersicon species revealed that the repeat is found in seven ancestral species of tomato.

No abstract

This result is discussed in terms of a higher order folding of viral DNA within the virus particle. Although the capsid architecture of the adenovirus particle is known in great detail, no fundamental understanding of the internal structure exists. Recent proposals for the structure of eukaryotic chromatin (1, 2) suggest a powerful model for the organization of the adenovirus core. The DNA in chromatin is wound around (3) specific histone complexes (4, 5) to form repeating subunits (6-8). The chromosomes of at least two animal viruses, polyoma and simian virus 40, are also condensed into subunits (9-11) within virus particles by complexing with cellular histones (12, 13). The adenovirus core does not contain histones, but we show in this paper that the core has a chromatin-like design. MATERIALS AND METHODSCell Culture and Synchronization. HeLa S3 cells were grown in suspension culture using medium F-13 (Grand Island Biological Co.) (17). The first two methods yielded "cornerless" particles which were not separated from free capsomers. Pyridine treatment stripped off the entire capsid. The resulting cores were further purified from capsomers by sediAbbreviation: bp, base pairs.* To whom reprint requests should be sent. mentation in a sucrose gradient. All preparations were adjusted to 1 mM CaCl2 and pH 7.5.Isolation of Nuclei. Cells were centrifuged at 500 X g for 5 min and resuspended at a concentration of 107 cells per ml in buffer containing 0.3 M sucrose, 10 mM Tris-cacodylate, 3 mM CaCl2, 0.5% Nonidet P-40, pH 7.8. Cells were lysed at 20 by repeatedly drawing (about 20 times) the suspension into a pasteur pipet. After 5 min, the nuclei were pelleted at 500 X g for 10 min and resuspended at 107 nuclei per ml in 0.3 M sucrose, 10 mM Tris-cacodylate, 1 mM CaCl2, pH 7.8.Nuclease Digestion. Staphylococcal nuclease (nucleate 3'-oligonucleotidohydrolase; EC 3.1.4.7) was purchased from Worthington Biochemical Corp. Reaction mixtures were incubated at 370 and reactions were terminated by adjusting the solutions to 10 mM EDTA, 1 mg of Pronase per ml, 2% Sarkosyl, and 10% glycerol. After 1 hr at 370, samples were applied directly to gels. One unit of nuclease corresponds to a change of 1 A260 unit at 37°.Gel Electrophoresis. Agarose (Sigma) gels containing 0.5 ,ug of ethidium bromide per ml were cast in 6 mm X 30 cm Plexiglas tubes. The running buffer, E buffer (18), contained 0.4 ,tg of ethidium bromide per ml. Electrophoresis was at 5 mA per gel for the indicated times. Gels were cut into 0.2 inch (0.5 cm) slices. Each slice was placed with 0.25 ml of H20 into 1 dram vials, autoclaved, and mixed with 2.5 ml of Aquasol. The radioactivity was determined by scintillation counting. RESULTS Nuclease digestion of disrupted adenovirus particlesWe have used staphylococcal nuclease (EC 3.1.4.7) to probe the structure of the adenovirus core. Adenovirus particles dialyzed against 5 mM Tris-maleate buffer at pH 6.4 lose pentons and neighboring hexons (17). The rest of the capsid, a shell of 180 hexons, still surrounds the...