Mutations That Improve the Binding of Yeast Flp Recombinase to Its Substrate

When yeast FLP recombinase is expressed from the phage lambda PR promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.     

Modulation of IMP dehydrogenase activity and guanylate metabolism by tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide)

Tiazofurin, a C-nucleoside, was cytotoxic in hepatoma 3924A cells grown in culture with an LC50 = 7.5 microM. In the culture, a closely linked dose-related response of tumor cell-kill and depletion of GTP pools was observed after tiazofurin treatment. In rats carrying subcutaneously transplanted hepatoma 3924A solid tumors, a single intraperitoneal injection of tiazofurin (200 mg/kg) caused a rapid inhibition of IMP dehydrogenase (EC 1.2.1.14) activity and depleted GDP, GTP, and dGTP pools in the tumor; concurrently, the 5-phosphoribosyl 1-pyrophosphate (PRPP) and IMP pools expanded 8- and 15-fold, respectively. Tiazofurin decreased tumoral IMP dehydrogenase activity and dGTP pools in a dose-dependent manner over a range of 50-200 mg/kg; by contrast, the depletion of GTP and the accumulation of IMP and PRPP pools were near maximum at 50 mg/kg. The increase in PRPP pools may be attributed to an inhibition by IMP of the activity of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8). The IMP dehydrogenase activity and the pools of ribonucleotides returned to the normal range by 24-48 h after the single injection of tiazofurin. However, the markedly depleted dGTP pools remained low for 72 h. Tiazofurin treatment resulted in significant anti-tumor activity in rats inoculated with hepatoma 3924A. The decrease in GTP levels and particularly the sustained depletion in the dGTP pools may explain, in part at least, the chemo-therapeutic action of tiazofurin on hepatoma 3924A. This is the first report showing that a marked therapeutic response was achieved against rapidly growing hepatoma 3924A by treatment with a single anti-metabolite.     

Substrate recognition by the 2 micron circle site-specific recombinase: effect of mutations within the symmetry elements of the minimal substrate.

The minimal substrate for the 2 microns circle site-specific recombinase FLP consists of a nearly perfect 13-base-pair dyad symmetry with an 8-base-pair core. By using a series of chemically synthesized FLP substrates in in vitro FLP recombination and FLP-binding assays, we have identified four positions within each of the symmetry elements that are important contact points for the FLP protein. Furthermore, the binding and recombination data provide evidence for cooperativity between the two symmetry elements of a substrate and between the symmetry elements of two partner substrates during FLP recombination.     

Grave's disease. Appearance in cytologic smears from fine needle aspirates of the thyroid gland

The cytomorphologic features were analyzed in thyroid samples obtained by fine needle aspiration (FNA) from 54 cases proven to be Grave's disease (toxic goiter) by a multiparameter study. A fire-flare appearance of the follicular cells and a good cellularity of the smears were the features most frequently observed in these cases. Hürthle cell changes were present in 47% of the cases, and small numbers of lymphocytes were seen in smears from 41% of the cases. Epithelioid cell granulomata and multinucleated giant cells were observed in less than one-fourth of the cases. About half of the cases showed some degree of pleomorphism of the follicular cells. Twenty cases of toxic multinodular goiter were similarly analyzed; the features that distinguished this entity from Grave's disease were the absence of epithelioid cells and multinucleated giant cells in the FNA smears.     

Fine needle aspiration cytologic diagnosis of the solitary cold thyroid nodule. Comparison with ultrasonography, radionuclide perfusion study and xeroradiography

Thirty-six cases of solitary and scintigraphically "cold" thyroid nodules were studied by fine needle aspiration (FNA) cytology, ultrasonography, radionuclide perfusion study (RPS) and xeroradiography with the aim of differentiating the neoplastic from the nonneoplastic nodules. Histologic study of the excised specimens provided the definitive diagnosis in all cases. Of the techniques used in this study, FNA cytology and RPS had the highest sensitivities and specificities. Ultrasonography and xeroradiography were of limited use due to their low sensitivity rates.     

Directed protein replacement in recombination full sites reveals trans-horizontal DNA cleavage by Flp recombinase.

One round of site-specific recombination between two DNA partners mediated by the Flp recombinase requires the breakage and reformation of four phosphodiester bonds. The reaction is accomplished by the combined action of four Flp monomers. Within the recombination complex, what is the relative disposition of a Flp monomer with respect to the target diester that it cleaves? To address this question, we have devised a strategy for the targeted orientation of Flp monomers within full-site recombination substrates. Our experimental design is not dependent on ''altered binding specificity'' of the recombinase. Analysis of the pattern of DNA cleavage by this method reveals no evidence for DNA cleavage in cis. A Flp monomer bound to its recognition element within the full site does not cleave the scissile phosphodiester bond adjacent to it. Our results are most consistent with ''trans-horizontal cleavage''. Cleavage by Flp occurs at the scissile phosphodiester distal to it, but within the same full site. The general experimental design employed here will be of widespread utility in mechanistic analyses of nucleic acid transactions involving multimeric DNA-protein assemblies.     

A domain sharing model for active site assembly within the Mu A tetramer during transposition: the enhancer may specify domain contributions.

The functional configuration of Mu transposase (A protein) is its tetrameric form. We present here a model for the organization of a functional Mu A tetramer. Within the tetramer, assembly of each of the two active sites for Mu end cleavage requires amino acid contributions from the central and C-terminal domains (domains II and III respectively) of at least two Mu A monomers in a trans configuration. The Mu enhancer is likely to function in this assembly process by specifying the two monomers that provide their C-terminal domains for strand cleavage. The Mu B protein is not required in this step. Each of the two active sites for the strand transfer reaction is also organized by domain sharing (but in the reverse mode) between Mu A monomers; i.e. a donor of domain II (also the recipient of domain III) during cleavage is a recipient of domain II (and the donor of domain III) during strand transfer. The function of the Mu B protein (which is required at the strand transfer step) and that of the enhancer element may be analogous in that their interactions with Mu A (domain III and domain I alpha respectively) promote conformations of Mu A conducive to strand cleavage or strand transfer.     

Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence.

The Flp protein catalyzes a site-specific recombination reaction between two 47 bp DNA sites without the assistance of any other protein or cofactor. The Flp recognition target (FRT) site consists of three nearly identical sequences, two of which are separated by an 8 bp spacer sequence. In order to gain insight into this remarkable protein-DNA interaction we used a variety of chemical probe methods and the missing nucleoside experiment to examine Flp binding. Hydroxyl radical footprints of Flp bound to a recombinationally-competent site fall on opposite faces of canonical B-DNA. The 8 bp spacer region between the two Flp binding sites becomes reactive towards 5-phenyl-1,10-phenanthroline.copper upon Flp binding, indicating that once bound by Flp, this segment of DNA is not in the B-form. Missing nucleoside analysis reveals that within each binding site the presence of two nucleosides on the top strand and four on the bottom, are required for formation of a fully-occupied FRT site. In contrast, loss of any nucleoside in the three binding sites in the FRT interferes with formation of lower-occupancy complexes. DNA molecules with gaps in the 8 bp spacer region are over-represented in complexes with either two or three binding sites occupied by Flp, evidence that DNA flexibility facilitates the cooperative interaction of Flp protomers bound to a recombinationally-active site.