A plant 3'-phosphoesterase involved in the repair of DNA strand breaks generated by oxidative damage.

Two novel, structurally and functionally distinct phosphatases have been identified through the functional complementation, by maize cDNAs, of an Escherichia coli diphosphonucleoside phosphatase mutant strain. The first, ZmDP1, is a classical Mg(2+)-dependent and Li(+)-sensitive diphosphonucleoside phosphatase that dephosphorylates both 3'-phosphoadenosine 5'-phosphate (3'-PAP) and 2'-PAP without any discrimination between the 3'- and 2'-positions. The other, ZmDP2, is a distinct phosphatase that also catalyzes diphosphonucleoside dephosphorylation, but with a 12-fold lower Li(+) sensitivity, a strong preference for 3'-PAP, and the unique ability to utilize double-stranded DNA molecules with 3'-phosphate- or 3'-phosphoglycolate-blocking groups as substrates. Importantly, ZmDP2, but not ZmDP1, conferred resistance to a DNA repairdeficient E. coli strain against oxidative DNA-damaging agents generating 3'-phosphate- or 3'-phosphoglycolate-blocked single strand breaks. ZmDP2 shares a partial amino acid sequence similarity with a recently identified human polynucleotide kinase 3'-phosphatase that is thought to be involved in DNA repair, but is devoid of 5'-kinase activity. ZmDP2 is the first DNA 3'-phosphoesterase thus far identified in plants capable of converting 3'-blocked termini into priming sites for reparative DNA polymerization.

Inhibition of RNA polymerase III elongation by a T10 peptide nucleic acid.

The terminator elements of eukaryotic class III genes strongly contribute to overall transcription efficiency by allowing fast RNA polymerase III (pol III) recycling. Being constituted by a run of thymidine residues on the coding strand (a poly(dA) tract on the transcribed strand), pol III terminators are expected to form highly stable triple-helix complexes with oligothymine peptide nucleic acids (PNAs). We analyzed the effect of a T10 PNA on in vitro transcription of three yeast class III genes (coding for two different tRNAs and the U6 small nuclear RNA) having termination signals of at least ten T residues. At nanomolar concentrations, the PNA almost completely inhibited transcription of supercoiled, but not linearized, templates in a sequence-specific manner. The total RNA output of the first transcription cycle was not affected by PNA concentrations strongly inhibiting multiple round transcription. Thus, an impairment of pol III recycling fully accounts for the observed inhibition. As revealed by the size and the state (free or transcription complex-associated) of the RNAs produced in PNA-inhibited reactions, pol III is "roadblocked" by the DNA-PNA adduct before reaching the terminator region. On different templates, the distance between the active site and the leading edge of the arrested polymerase ranged from 10 to 20 base pairs. Given their ability to efficiently block pol III elongation, oligothymine PNAs lend themselves as potential cell growth inhibitors interfering with eukaryotic class III gene transcription.

TFIIIC-independent in vitro transcription of yeast tRNA genes.

The most peculiar transcriptional property of eukaryotic tRNA genes, as well as of other genes served by RNA polymerase III, is their complete dependence on the intragenic interaction platform provided by transcription factor IIIC (TFIIIC) for the productive assembly of the TBP-containing initiation factor TFIIIB. The sole exception, in yeast, is the U6 RNA gene, which is able to exploit a TATAAATA element, 30 bp upstream of the transcription start site, for the TFIIIC-independent assembly of TFIIIB. To find out whether this extragenic core promoter organization and autonomous TFIIIB assembly capacity are unique features of the U6 gene or also apply to other genes transcribed by RNA polymerase III, we scanned the 5'-flanking regions (up to position -100) of the entire tRNA gene set of Saccharomyces cerevisiae searching for U6-like TATA motifs. Four tRNA genes harboring such a sequence motif around position -30 were identified and found to be transcribed in vitro by a minimal system only composed of TFIIIB and RNA polymerase III. In this system, start site selection is not at all affected by the absence of TFIIIC, which, when added, significantly stimulates transcription by determining an increase in the number, rather than in the efficiency of utilization, of productive initiation complexes. A specific TBP-TATA element interaction is absolutely required for TFIIIC-independent transcription, but the nearby sequence context also contributes to the efficiency of autonomous TFIIIB assembly. The existence of a TFIIIB assembly pathway leading to the faithful transcription of natural eukaryotic tRNA genes in the absence of TFIIIC provides novel insights into the functional flexibility of the eukaryotic tRNA gene transcription machinery and on its evolution from an ancestral RNA polymerase III system relying on upstream, TATA- centered control elements.

tRNA-assisted overproduction of eukaryotic ribosomal proteins.

Structural studies of eukaryotic ribosomes are complicated by the tendency of their constituent proteins to be expressed at very low levels in Escherichia coli. We find that this is mainly due to their exceptionally high content of AGA/AGG arginine codons, which are poorly utilized by the bacterial translational machinery. In fact, we could overcome this limitation by the combined use of a T7 RNA polymerase expression vector and a plasmid carrying the E. coli gene argU, which encodes the minor tRNA(Arg) species that reads AGA/AGG codons. In this system, five cytoplasmic ribosomal proteins from three different eukaryotic lineages (Saccharomyces cerevisiae S8, L13, and L14; Arabidopsis thaliana L13; and Homo sapiens L7) could be overexpressed to up to 50% of total bacterial protein and were purified to homogeneity in tens of milligrams amounts. The purification procedure simply involved metal affinity chromatography followed, in some cases, by an additional heparin chromatography step. Recombinant polypeptides bound RNA with high affinity (K(d) between 50 and 300 nM). This novel overexpression/purification strategy will allow the production of high amounts of most eukaryotic ribosomal proteins in a form suitable for structural and functional studies. Coupled with recently completed and ongoing whole-genome sequencing projects, it will facilitate the molecular characterization of the eukaryotic ribosome.

Domain organization and functional properties of yeast transcription factor IIIA species with different zinc stoichiometries.

Transcription factor IIIA (TFIIIA) binds to the 5 S rRNA gene through its zinc finger domain and directs the assembly of a multiprotein complex that promotes transcription initiation by RNA polymerase III. Limited proteolysis of TFIIIA forms with different zinc stoichiometries, in combination with DNA binding and in vitro transcription analyses, have been used herein to investigate the domain organization and zinc requirements of Saccharomyces cerevisiae TFIIIA. Species containing either nine, six, or three zinc equivalents were produced by reductive resaturation and controlled metal depletion of recombinant TFIIIA. Partial digestion of the metal-saturated, 9 Zn2+-liganded factor yields a stable intermediate comprising the eight N-terminal zinc fingers, and a less stable fragment corresponding to a C-terminal portion including the ninth finger. Proteolyzed TFIIIA has the same 5 S DNA binding ability of the intact protein yet no longer supports in vitro 5 S rRNA synthesis. Both the structural compactness and the 5 S DNA binding ability of the TFIIIA form only containing 3 zinc ions are severely compromised. In contrast, the 6 Zn2+-liganded species was found to be indistinguishable from metal-saturated TFIIIA. By demonstrating the existence of three classes of zinc-binding sites contributing differently to yeast TFIIIA structure and function, the present study provides new evidence for the remarkable flexibility built into this complex transcription factor.

Functional interchangeability of TFIIIB components from yeast and human cells in vitro.

In eukaryotes, TFIIIB is required for proper initiation by RNA polymerase III. In the yeast Saccharomyces cerevisiae a single form of TFIIIB (gammaTFIIIB) is sufficient for transcription of all pol III genes, whereas in extracts derived from human cells two different hTFIIIB complexes exist which we have previously designated as hTFIIIB-alpha and hTFIIIB-beta. Human TFIIIB-alpha is a TBP-free entity and must be complemented by TBP for transcription of pol III genes driven by gene external promoters, whereas hTFIIIB-beta is a TBP-TAF complex which governs transcription from internal pol III promoters. We show that hTFIIIB-beta cannot be replaced by yeast TFIIIB for transcription of tRNA genes, but that the B" component of gammaTFIIIB can substitute for hTFIIIB-alpha activity in transcription of the human U6 gene. Moreover, hTFIIIB-alpha can be chromatographically divided into activities which are functionally related to gammaTFIIIE and recombinant yB"90, suggesting that hTFIIIB-alpha is a human homolog of yeast TFIIIB". In addition, we show that yeast TBP can only be exchanged against human TBP for in vitro transcription of the human and yeast U6 gene but virtually not for that of the yeast tRNA4Sup gene. This deficiency can be counteracted by a mutant of human TBP (R231K) which is able to replace yeast TBP for transcription of yeast tRNA genes in vitro.

Steroid-eluting electrodes prevent chronic pacing threshold rise in the atrial chamber after oral propafenone administration.

The aim of the study was to evaluate chronic atrial pacing threshold increase after oral propafenone therapy. Fifty patients affected by advanced AV block and sick sinus syndrome were studied at least 6 months after pacemaker implantation, before and after oral propafenone therapy (450-900 mg/day based on body weight). The patients were subdivided into three groups as to the type of electrode implanted, all three unipolar: group I (20 patients) Medtronic CapSure 4003, group II (13 patients) Medtronic Target Tip 4011, group III (17 patients) Medtronic 4057 screw-in leads. In all cases, Medtronic unipolar pacemakers were implanted with the same noninvasive autothreshold measurement method. Propafenone and 5-OH-propafenone blood levels were measured 3-5 hours after drug administration. The packing autothreshold was measured at 0.8, 1.6, and 2.5 V by reducing the pulse width. After propafenone, groups II and III showed a statistically significant threshold rise (P ranging from < 0.01 to 0.05), whereas no significant difference was found in group I. Propafenone and 5-OH-propafenone blood vessels did not show any significant difference among the three groups. Strength-duration curves were drawn for the three groups before and after propafenone: at baseline the curves shifted to the left with the steep part above the knee, clearly favoring CapSure over the other two groups. After propafenone, the curves shifted to the right, with the flat par progressively more evident in groups II and III. In the atrial chamber, steroid-eluting leads prevented threshold increase after propafenone therapy, in contrast with a significant threshold rise with conventional porous and screw-in leads.

A suppressor of mutations in the class III transcription system encodes a component of yeast TFIIIB.

Class III genes depend on TFIIIB for recruitment of RNA polymerase III. Yeast TFIIIB is comprised of three components: TBP, TFIIIB70 and a 90 kDa polypeptide contained in the fraction B". We report the isolation of the yeast gene TFC7 which, based on genetic and biochemical evidence, encodes the 90 kDa polypeptide. TFC7 was isolated as a multicopy suppressor of temperature-sensitive mutations in the two largest subunits of TFIIIC. It is an essential gene, encoding a polypeptide of 68 kDa migrating with an apparent size of approximately 90 kDa. In gel shift assays, recombinant TFC7 protein (rTFC7) alone did not bind detectably to DNA, or to the TFIIIC-DNA complex even in the presence of TBP or TFIIIB70, but it was required to assemble the TFIIIB-TFIIIC-DNA complex. The two-hybrid assay pointed to an interaction between TFC7 protein and tau 131, the second largest subunit of TFIIIC (that also interacts with TFIIIB70). rTFC7p can replace the B" component of TFIIIB for synthesis of U6 RNA in a system reconstituted with recombinant TBP and TFIIIB70 polypeptides and highly purified RNA polymerase III. Surprisingly, specific transcription of the SUP4 tRNATyr gene promoted by rTFC7p was much weaker than with B". An additional factor activity, provided by the recently identified TFIIIE fraction, was required to restore control levels of transcription.

Facilitated recycling pathway for RNA polymerase III.

We show that the high in vitro transcription efficiency of yeast RNA pol III is mainly due to rapid recycling. Kinetic analysis shows that RNA polymerase recycling on preassembled tDNA.TFIIIC.TFIIIB complexes is much faster than the initial transcription cycle. High efficiency of RNA pol III recycling is favored at high UTP concentrations and requires termination at the natural termination signal. Runoff transcription does not allow efficient recycling. The reinitiation process shows increased resistance to heparin as compared with the primary initiation cycle, as if RNA polymerase was not released after termination. Indeed, template competition assays show that RNA pol III is committed to reinitiate on the same gene. A model is proposed where the polymerase molecule is directly transferred from the termination site to the promoter.

A universally conserved region of the largest subunit participates in the active site of RNA polymerase III.

The largest subunits of the three eukaryotic nuclear RNA polymerase present extensive sequence homology with the beta' subunit of the bacterial enzymes over five major co-linear regions. Region d is the most highly conserved and contains a motif, (Y/F)NADFDGD(E/Q)M(N/A), which is invariant in all multimeric RNA polymerases. An extensive mutagenesis of that region in yeast RNA polymerase III led to a vast majority (16/22) of lethal single-site substitutions. A few conditional mutations were also obtained. One of them, rpc160-112, corresponds to a double substitution (T506I, N509Y) and has a slow growth phenotype at 25 degrees C. RNA polymerase III from the mutant rpc160-112 was severely impaired in its ability to transcribe a tRNA gene in vitro. The transcription defect did not originate from a deficiency in transcription complex formation and RNA chain initiation, but was mainly due to a reduced elongation rate. Under conditions of substrate limitation, the mutant enzyme showed increased pausing at the intrinsic pause sites of the SUP4 tRNA gene and an increased rate of slippage of nascent RNA, as compared with the wild-type enzyme. The enzyme defect was also detectable with poly[d(A-T)] as template, in the presence of saturating DNA, ATP and UTP concentrations. The mutant enzyme behavior is best explained by a distortion of the active site near the growing point of the RNA product.

Selective inactivation of two components of the multiprotein transcription factor TFIIIB in cycloheximide growth-arrested yeast cells.

Following protein synthesis inhibition in cycloheximide growth-arrested yeast cells, the rates of tRNA and 5 S RNA synthesis decrease with apparent half-times of about 20 and 10 min, respectively. This effect is mimicked by extracts of treated cells, and the impairment of tRNA gene transcription activity that is observed in vitro parallels the in vivo inactivation of RNA polymerase III transcription. As revealed by experiments in which partially purified class III transcription factors were singly added to extracts of treated cells, only the activity of the multiprotein transcription factor TFIIIB is severely impaired after 3 h of cycloheximide treatment. Similar assays carried out in an in vitro transcription system in which TFIIIB activity was reconstituted by a combination of the TATA box-binding protein (TBP), the 70-kDa component TFIIIB70, plus a partially purified fraction known as B" have shown that the latter two components are both necessary and sufficient to restore control levels of transcription. Their activity, but not TBP activity, is considerably reduced in extracts of treated cells. TFIIIB70 and a component of fraction B" thus appear to be the selective targets of the down-regulation of polymerase III transcription that is brought about by cycloheximide. A substantial depletion of the TFIIIB70 polypeptide was detected by Western immunoblot analysis of extracts derived from cycloheximide growth-arrested cells, indicating that the inactivation of this TFIIIB component results primarily from its enhanced destabilization under conditions of protein synthesis inhibition.

High level expression in E. coli and purification of yeast transcription factor IIIA.

Saccharomyces cerevisiae transcription factor IIIA, a sequence-specific DNA binding protein that is required for transcription of 5S rRNA genes by RNA polymerase III, has been expressed in Escherichia coli in a full length, native form. High level expression was achieved through the combined use of a T7 RNA polymerase expression system and of a multicopy plasmid carrying an E. coli gene, argU, which codes for a minor Arg(AGA/AGG) tRNA species. Recombinant yeast transcription factor IIIA was purified to 95% homogeneity, at a final yield of 8 mg/liter of bacterial culture, by three chromatographic steps, and it was shown to be at least 55% active by quantitative in vitro transcription assays.

Identification of new eukaryotic tRNA genes in genomic DNA databases by a multistep weight matrix analysis of transcriptional control regions.

A linear method for the search of eukaryotic nuclear tRNA genes in DNA databases is described. Based on a modified version of the general weight matrix procedure, our algorithm relies on the recognition of two intragenic control regions known as A and B boxes, a transcription termination signal, and on the evaluation of the spacing between these elements. The scanning of the eukaryotic nuclear DNA database using this search algorithm correctly identified 933 of the 940 known tRNA genes (0.74% of false negatives). Thirty new potential tRNA genes were identified, and the transcriptional activity of two of them was directly verified by in vitro transcription. The total false positive rate of the algorithm was 0.014%. Structurally unusual tRNA genes, like those coding for selenocysteine tRNAs, could also be recognized using a set of rules concerning their specific properties, and one human gene coding for such tRNA was identified. Some of the newly identified tRNA genes were found in rather uncommon genomic positions: 2 in centromeric regions and 3 within introns. Furthermore, the presence of extragenically located B boxes in tRNA genes from various organisms could be detected through a specific subroutine of the standard search program.

A novel RNA polymerase III transcription factor fraction that is not required for template commitment.

We have identified and partially characterized a novel class III transcription factor fraction (TFIIIE) from yeast nuclear extracts. TFIIIE is functionally distinct from the standard yeast transcription factor fractions, TFIIIB and TFIIIC. It is also different from either of the TFIIIB subfractions, B' and B". TFIIIE is essential for specific transcription of both tRNA and 5 S RNA genes, its activity is sensitive to proteinase K, and it exhibits an apparent sedimentation coefficient of 4.0 S when analyzed on glycerol gradients. In the case of a tRNA gene, TFIIIE does not play a role in the formation of stable preinitiation complexes containing TFIIIB and TFIIIC. It is required for single as well as multiple rounds of transcription, however. Thus, TFIIIE is involved in the utilization of stable transcription complexes, but its action is not restricted to reinitiation events.

[The high-resolution analysis of the P wave recorded via the esophagus: a new diagnostic approach in patients with paroxysmal atrial fibrillation]. / L'analisi ad alta risoluzione dell'onda P registrata per via transesofagea: nuovo approccio diagnostico in pazienti con fibrillazione atriale parossistica.

BACKGROUND: P-wave signal averaged ECG has recently been proposed in the evaluation of patients with Paroxysmal Atrial Fibrillation (PAF). The aim of this study was to verify the utility of this method in the characterisation of these patients utilizing a transoesophageal signal. METHODS: The study population consisted of two groups of subjects: Group A, 34 consecutive patients (16 males; 57 +/- 9 yrs) with a documented PAF and without any underlying heart disease, and Group B, normal controls (9 males; 44 +/- 6 yrs). A signal-averaged ECG, triggered with the oesophageal P-wave, obtained from an X, Y, Z lead system was recorded in each patient, utilizing the AEROTEL HIPEC 200 system. The three orthogonal averaged ECGs were band-pass filtered (non-recursive filter, 40-250 Hz) and combined into a spatial vector magnitudo. We evaluated the filtered P-wave duration (Ad, msec) and the Root Mean Square (RMS) voltages of the last 10, 20, and 30 msec of the P vector magnitudo (RMS 10, 20, 30; mcV). RESULTS: The patients with PAF presented with a significantly longer Ad duration (A 126.6 +/- 12.4 vs B 96.8 +/- 12.5 msec, < 0.001), and lower amplitudes of RMS 10, 20, 30 (RMS 10 A 4.8 +/- vs B 9 +/- 2.7 p < 0.005; RMS 20 A 6.9 +/- 2.3 vs B 15.7 +/- 7.3 p < 0.005; RMS30 A 12.1 +/- 5.1 vs B 25.3 +/- 11.5 p < 0.005). An Ad > or = 100 msec, a value of RMS 10 < or = 6.5, RMS 20 < or = 9 and RMS 30 < or = 12.5 mcV showed high values of sensitivity, specificity and positive predictive value (Ad 85%, 100%, 100%, RMS10 93%, 80%, 90%, RMS20 84%, 90%, 96%, RMS30 72%, 90%, 72%). The combined duration/voltage criteria (Ad +/- 110 msec and RMS10 < or = 6.5 mcV or RMS20 < or = 9 mcV) showed a sensitivity of 80% and 76% with a specificity and positive predictive value of 100%. CONCLUSIONS: The P-wave triggered transoesophageal atrial signal averaged ECG appears able to identify patients with PAF; however, prospective studies must investigate the possible clinical use of these findings.

Cardiac pacing in unilateral left superior vena cava: evaluation by digital angiography.

In a patient with complete heart block and chronic lymphocytic leukemia a pacemaker lead could not be introduced from either the right or left subclavian vein. Digital subtraction angiography excluded a neoplastic mediastinal mass, demonstrated a unilateral left superior vena cava and defined the best route for lead insertion.

[Continent cecal-colonic reservoir. Surgical technique]. / Serbatoio cieco-colico continente. Technica operatoria.

The Authors discuss a recent case report treated with radial cystectomy associated with a secondary urinary derivation using the caecum-colon reservoir. After having reviewed the various surgical procedures involving the urinary derivations, the Authors describe the technique used by them paying particular attention to the positive aspects of having a low filling pressure reservoir controlled by a valid sphincter ileum-caecum valve. Considering the good postoperative result with this method, the Authors regard this procedure as an alternative to other urinary derivation techniques when carried out with correct indications.

[The efficacy of oral treatment with flecainide for paroxysmal atrial fibrillation: correlation with plasma concentration]. / Efficacia del trattamento orale con flecainide nella fibrillazione atriale parossistica: correlazione con le concentrazioni plasmatiche.

In the acute treatment of paroxysmal atrial fibrillation several drugs can be used. The aim of our work was to assess the efficacy of a single oral dose of flecainide in the conversion to sinus rhythm by correlating this data with flecainide plasma concentration. We have considered 37 patients affected by paroxysmal atrial fibrillation (for more than 8 hours) randomly assigned to the following two groups: group A, 19 patients, mean age 44.4 +/- 1.9 years) treated with flecainide (200 mg) and control group B (18 patients, mean age 46.6 +/- 1.8 years). This was done in order to point out any possible overlap between pharmacological and spontaneous conversion to sinus rhythm. In all patients, the following were performed: a Holter recording (524 hours) to evaluate the time of conversion to sinus rhythm (t-conversion to sinus rhythm), a determination of flecainide plasma concentration (after 150 flecainide administration) an Rx, an Echo-2D/Doppler test and an estimation of thyroid function. The Rx, the Echo-2D/Doppler and the endocrinological data in the 2 groups did not show any significant differences. We obtained a conversion to sinus rhythm in all but one of the group A patients (time of conversion to sinus rhythm 162 +/- 83 min) and in just 5 group B patients (time of conversion to sinus rhythm 1118 +/- 125 min) (time of conversion to sinus rhythm A vs B p less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)