Ribosomal frameshifting regulates expression of the TYB gene of yeast Ty retrotransposons. We previously demonstrated that a 14 nucleotide sequence conserved between two families of Ty elements was necessary and sufficient to support ribosomal frameshifting. This work demonstrates that only 7 of these 14 nucleotides are needed for normal levels of frameshifting. Any change to the sequence CUU-AGG-C drastically reduces frameshifting; this suggests that two specific tRNAs, tRNA(UAGLeu) and tRNA(CCUArg), are involved in the event. Our tRNA overproduction data suggest that a leucyl-tRNA, probably tRNA(UAGLeu), an unusual leucine isoacceptor that recognizes all six leucine codons, slips from CUU-Leu onto UUA-Leu (in the +1 reading frame) during a translational pause at the AGG-Arg codon induced by the low availability of tRNA(CCUArg), encoded by a single-copy essential gene. Frameshifting is also directional and reading frame specific. Interestingly, frameshifting is inhibited when the "slip" CUU codon is located three codons downstream, but not four or more codons downstream, of the translational initiation codon.

The Ty (transposon yeast) family of retroviral-like transposons include two genes, TYA and TYB, analogous to the gag andpol genes ofmetazoan retroviruses. TYB lies downstream of TYA, the two genes overlapping by 38 base pairs. The primary translation product of TYB is a TYA/TYB fusion protein whose expression has been inferred to occur by translational frameshifting within the overlap region. We show that the event leading to expression of TYB is very efficient, resulting in 20% read-through into TYB from TYA. We demonstrate that the Ty mRNA is colinear with the DNA sequence of the element, eliminating any pretranslational model for TYB expression. Frameshifting requires no particular sequence of the upstream TYA gene, nor any global RNA structure. Surprisingly, it can be promoted by a 14-base-pair oligonucleotide of the overlap region. The ability of this oligonucleotide to function is inhibited when it is positioned immediately downstream of an initiator AUG. We conclude that the TYB gene is expressed by an efficient ribosomal frameshifting event requiring a small oligonucleotide sequence derived from the TYAITYB overlap region.The Ty (transposon yeast) elements of the yeast Saccharomyces cerevisiae are a family of about 30 dispersed retroviral-like transposons (1, 2). Ty elements include two open reading frames, termed TYA and TYB (3), which are analogous respectively to the gag and pol genes of metazoan retroviruses. The similarity in structure and organization reflects the fact that Ty elements, like retroviruses, replicate via an RNA intermediate during transposition (2). The primary translation product of retroviral pol genes and TYB are fusions to the product ofthe upstream gene, gag and TYA. In the case of most of the avian and mammalian retroviruses the gag, pol, and sometimes pro (encoding retroviral protease) genes overlap; similarly TYB overlaps the last 38-44 base pairs (bp) of TYA. For Rous sarcoma virus (RSV) (4) and mouse mammary tumor virus (MMTV) (5, 6), translational frameshifting has been shown explicitly to produce the fusion peptides. It is likely, given their similarity to retroviruses, that Ty elements also express theirpol analog by translational frameshifts; however, the evidence for this is circumstantial. Nuclease S1 protection experiments eliminate the possibility that TYB expression involves RNA splicing (3,7,8). The experiments are sufficiently sensitive to detect a very small discontinuity between the RNA and the DNA [5-11 nucleotides (nt)]; these experiments do not exclude the possibility that expression of TYB occurs by RNA editing.Wilson et al. (8) have demonstrated that a 125-bp region of the element Tyl-15, extending from a position 7 bp into the overlap through a Bgl II site 94 bp downstream ofthe overlap, is sufficient to promote normal TYAITYB expression. They also showed that a deletion removing the first 24 nt of the overlap eliminates TYAITYB expression. They speculated that a short region of the overlap that is conserved between the TyJ and Ty2 class elements,...

Ribonucleotide reductase catalyzes the production of deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates for DNA synthesis. Mammalian ribonucleotide reductase (RNR) is a tetramer consisting of two non-identical homodimers, R1 and either R2 or p53R2, which are considered to be involved in DNA replication and repair, respectively. We have demonstrated that DNA damage by doxorubicin and cisplatin caused a steady elevation of the R2 protein in p53(؊/؊) HCT-116 human colon carcinoma cells but induced degradation of the protein in p53(؉/؉) cells. To evaluate the involvement of R2 in response to DNA damage, p53(؊/؊) HCT-116 cells were stably transfected with an expression vector transcribing short hairpin/short interference RNA directed against R2 mRNA. Stably transfected clones exhibited a pronounced reduction of the R2 protein with no change in the cellular growth rate. Furthermore, short interference RNA-mediated reduction of the R2 protein caused a marked increase in sensitivity to the DNA-damaging agent cisplatin as well as to the RNR inhibitors Triapine® and hydroxyurea. Ectopic expression of p53R2 partially reversed the cytotoxicity of cisplatin but not that of RNR inhibitors to R2 knockdown cells. The increase in sensitivity to cisplatin and RNR inhibitors was correlated with the suppression of dATP and dGTP levels caused by stable expression of R2-targeted short interference RNA. These results indicated that DNA damage resulted in elevated levels of the R2 protein and dNTPs and, consequently, enhanced the survival of p53(؊/؊) HCT-116 cells. The findings provide evidence that R2-RNR can be employed to supply dNTPs for the repair of DNA damage in cells with an impaired p53-dependent induction of p53R2. Ribonucleotide reductase (RNR)1 catalyzes a rate-limiting reaction in which ribonucleoside diphosphates are converted to their corresponding deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates (dNTPs) required for DNA synthesis and repair (1). In mammalian cells, the catalytically active RNR is considered to be an ␣ 2 ␤ 2 heterotetramer consisting of two large R1 subunits and two small R2 subunits. To maintain a balanced size of dNTP pools (2), the enzymatic activity of RNR is tightly regulated by the binding of nucleoside triphosphates (ATP, dATP, dGTP, and dTTP) to allosteric sites in the R1 subunit (1). In proliferating cells, the level of the R2 protein is low in the G 1 phase of the cell cycle but accumulates and reaches maximal levels during the S phase, followed by degradation when passing through the G 2 /M phase (3-6). Thus, the activity of RNR is additionally controlled by cell cycle-specific availability of the R2 subunit, whereas the R1 protein level remains relatively constant throughout the cell cycle (3, 4). When cells undergo G 1 arrest following DNA damage, the supply of dNTPs for DNA repair is, in turn, provided by transcriptional activation of the recently identified R2 homologue, p53R2 (7, 8). Thus, DNA damage causes p53-depend...

The study was designed to evaluate whether TAPET-CD, an attenuated strain of Salmonella typhimurium ex pressing Escherichia coli cytosine deaminase (CD), was capable of converting nontoxic 5-fluorocytosine (5-FC) to the active antitumor agent 5-fluorouracil (5-FU). The antitumor effect of TAPET-CD plus 5-FC against subcutaneously implanted colon tumors was also evaluated. TAPET-CD was given to tumor-bearing mice by a single bolus intravenous administration followed with 5-FC by intraperitoneal administration. TAPET-CD accumulated in tumors at levels 1000-fold higher than that in normal tissues and high levels of 5-FU were de growth by 88%-96%, compared to TAPET-CD alone, which inhibited tumor growth by 38%-79%. These data suggest that tumor-targeting Salmonella could be used to deliver prodrug-converting enzyme selectively to tumors and produced anti-tumor effects when the corresponding prodrug was also given. These studies demonstrate the potential use of attenuated Salmonella as a tumor-selective protein delivery vector.

Purified NADPH:cytochrome c (P-450) reductase (FPT; NADPH-ferrihemoprotein oxidoreductase, EC (for appropriate references, see refs. 9-11). Evidence exists to suggest that alkylation of DNA is the critical lesion in the cytotoxicity of the mitomycins to both hypoxic and oxygenated cells (12, 13).Studies in mice bearing transplanted solid tumors demonstrated the ability of these antibiotics to reach and kill hypoxic cells and, in combination with x-irradiation, to produce additive or greater than additive cytotoxicity to tumor cells without a corresponding increase in toxicity to normal tissues (10,11

Some 4- and 2-(nitrobenzyloxycarbonyl)-1, 2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines (4, 6, and 7) were synthesized and evaluated for their ability to exert preferential toxicity to hypoxic EMT6 mammary carcinoma cells using a colony-forming assay. Of these, the 4,5-dimethoxy-2-nitro analogue 6 (50 microM, 1-h exposure) caused greater than 3 logs of kill of hypoxic cells, with relatively minor toxicity to corresponding aerobic cells. The ability of 4-nitro (4) and 4,5-dimethoxy-2-nitro (6) analogues to reach and kill hypoxic cells of solid tumors was also demonstrated using intradermally implanted EMT6 solid tumors in mice. In addition, a possible source of toxicity to normal tissue, i. e., the activation of the 4-nitrobenzyl derivative 4 by glutathione S-transferase-catalyzed thiolysis, was essentially eliminated by replacing one of the benzylic methylene protons by a methyl group. The 4-nitro (4) and 4,5-dimethoxy-2-nitro (6) analogues also appear to be reduced more easily under acidic conditions (pH 6.0) than under neutral conditions, as measured by differential pulse polarography. Since the pH in hypoxic regions is often lower than that in adjacent aerobic regions, this property should aid in the cytotoxic action of these agents against hypoxic cells of solid tumors.

Treatment of EMT6 mouse mammary tumor cells with mitomycin C (MC) results in the formation of six major MC−DNA adducts. We identified the last unknown of these (“adduct X”) as a guanine N2 adduct of 2,7-diaminomitosene (2,7-DAM), in which the mitosene is linked at its C-10 position to guanine N2. The assigned structure is based on UV and mass spectra of adduct X isolated directly from the cells, as well as on its difference UV, second-derivative UV, and circular dichroism spectra, synthesis from [8-3H]deoxyguanosine, and observation of its heat stability. These tests were carried out using 17 μg of synthetic material altogether. The mechanism of formation of adduct X involves reductive metabolism of MC to 2,7-DAM, which undergoes a second round of reductive activation to alkylate DNA, yielding adduct X and another 2,7-DAM−guanine adduct (adduct Y), which is linked at guanine N7 to the mitosene. Adduct Y has been described previously. Adduct X is formed preferentially at GpC, while adduct Y favors the GpG sequence. In contrast to MC−DNA adducts, the 2,7-DAM−DNA adducts are not cytotoxic.

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