Transcription in vitro of ø29 DNA and EcoRI fragments by Bacillus subtilis RNA polymerase

EcoRI fragments A, B and C produced from linear φ29 DNA, but not D or E fragments, are transcribed by purified Bacillus subtilis RNA polymerase. The transcription of fragments A and C is initiated preferentially with GTP and to a lesser extent with ATP; the reverse happens in the case of fragment B. The dinucleotides GpU and GpA respectively, compete specifically with the incorporation of [γ-32P]GTP directed by fragments A and C. The RNA synthesized in vitro by purified B. subtilis RNA polymerase is highly asymmetric. Most of the RNA synthesis directed by fragments A and C is early RNA. However, most of the RNA produced by fragment B is anti-late-RNA. Addition of crude extracts inhibit the transcription of fragment B but not that of fragments A and C.Comisión Asesora para el Desarrollo de la Investigación Científica y Técnica y Dirección General de SanidadPeer reviewe

Isolation of a strong suppressor of nonsense mutations in Bacillus subtilis

By treatment of Bacillus subtilis MO-101-P spoA− met thr− su− with ethyl methanesulfonate, a strong suppressor strain of nonsense mutations, B. subtilis MO-101-P spoA− [met−]+ thr− su+44 was isolated. This strain does not suppress phage φ 29 mutant susB47, selected on a B. subtilis strain containing the su+3 suppressor isolated by Georgopoulos. A revertant from this mutant, susB610, was isolated, being suppressed by both the su+3 and su+44 suppressor strains. The efficiency of suppression by strain su+44 is about 50%. The experiments shown in this paper suggest that strain su+44 contains an amber and strain su+3 an ochre suppressor.This investigation has been aided by grants from Comisión Asesora para el Drsarrollo de la Investigacion Cientifica y Tecnica, Comision del Descuenio Complr- rnentario (I.N.P.) and Dirección General de Sanidad. R. P .M. is a Fellow of Fondo Nacional para la Formación de Personal Investigador.Peer reviewe

The African Swine Fever Virus IAP Homolog Is a Late Structural Polypeptide

AbstractThe analysis of the complete nucleotide sequence of the African swine fever virus genome has revealed the existence of a number of genes potentially capable of modifying the host's response to the virus infection. In this report, we describe the results of the characterization of the A224L gene that encodes a novel member of the family of apoptosis inhibitors known as IAP proteins. A224L is expressed during the late phase of the infectious cycle, and its polypeptide product is assembled into virus particles

African swinw fever (ASF) virus: A missing link between poxviruses and iridoviruses

Trabajo presentado en el VII International Congress of Virology, celebrado en Edmonton (Canadá) del 09 al 14 de agosto de 1987

Temperature-sensitive mutants affected in DNA synthesis in phage ø29 of Bacillus subtilis

The DNA synthesis in Bacillus amyloliquefaciens infected with wild-type phage ϕ29 and with temperature-sensitive mutants has been studied in the presence of the drug 6-(p-hydroxy-phenylazo)-uracil. The drug inhibits DNA synthesis in uninfected bacteria while it does not affect DNA synthesis and phage development in ϕ29-infected bacteria. DNA synthesis was blocked by infection at the restrictive temperature with the mutants F99 and K132, representatives of the two early functions identified so far, as well as with mutant C17, which affects a late function. The remaining mutants, belonging to eight complementation groups, synthesized DNA at the restrictive temperature. The proportion of the DNA resistant to DNAase was different, depending on the mutant.This investigation has been aided by a Grant from the Jane Coffin Childs Memorial Fund for Medical Research. A. T. is a fellow of Pond0 Nacional para la Formacidn de Personal Investigador.Peer reviewe

Isolation of a strong suppressor of nonsense mutations in Bacillus subtilis

By treatment of Bacillus subtilis MO-101-P spoA− met thr− su− with ethyl methanesulfonate, a strong suppressor strain of nonsense mutations, B. subtilis MO-101-P spoA− [met−]+ thr− su+44 was isolated. This strain does not suppress phage φ 29 mutant susB47, selected on a B. subtilis strain containing the su+3 suppressor isolated by Georgopoulos. A revertant from this mutant, susB610, was isolated, being suppressed by both the su+3 and su+44 suppressor strains. The efficiency of suppression by strain su+44 is about 50%. The experiments shown in this paper suggest that strain su+44 contains an amber and strain su+3 an ochre suppressor.This investigation has been aided by grants from Comisión Asesora para el Drsarrollo de la Investigacion Cientifica y Tecnica, Comision del Descuenio Complr- rnentario (I.N.P.) and Dirección General de Sanidad. R. P .M. is a Fellow of Fondo Nacional para la Formación de Personal Investigador.Peer reviewe

Synthesis in vitro of Ψ29-Specific Early Proteins Directed by Phage DNA

The RNA and proteins synthesized in an Escherichia coli cell-free system of protein synthesis directed by Bacillus subtilis phage Ψ29 DNA were studied. Hybridization-competition experiments showed that most of the RNA species synthesized in vitro are early RNAs. Many of the early proteins induced after phage infection were also synthesized in the E. coli cell-free system. None of the late proteins, structural or non-structural, were synthesized in the system in vitro.This investigation has been aided by a grant from the Comision Asesora para el Desarrollo de la investigacion Cientifica. J. L. C. and F. J. are Fellows of Fondo Nacional para lu Formacion de Personal Investigador.Peer reviewe

Induced biosynthesis of liver glucokinase

A glucokinase qualitatively and quantitatively adequate as the first and limiting step in the synthesis of glycogen from glucose in liver has recently been identified in normal fed rats. In contrast with hexokinase, that also occurs in liver, glucokinase activity disappears in fasted and alloxandiabetic animals. The high Km, glucose-6-P-insensitive glucokinase, does not occur in muscle, nor in any of several other tissues examined. This and other indirect evidence suggest that the glucokinase is a unique enzyme of the parenchymal cells of the liver, and that the hexokinase measured in liver homogenates is present in the non-parenchymal cells. Glucokinase reappears in diabetic and fasted animals within a few hours after administration of insulin or refeeding respectively. Glucose administration alone is also effective in provoking the reappearance of glucokinase activity in previously fasted rats. Liver hexokinase remains essentially unaltered in these conditions. Inhibition of the reappearance of glucokinase by ethionine and p-fluorophenylalanine, largely counteracted by methionine and phenylalanine respectively, suggests that actual synthesis of the enzyme is involved. A similar effect of actinomycin supports this conclusion while it further suggests that formation of messenger RNA is involved. These results suggest that liver glucokinase is an inducible enzyme whose synthesis is induced directly or indirectly, by insulin. The differences in the hexosemonophosphate pathway between liver and muscle, their metabolic regulation, and the relationship with other pathways is discussed.Supported by a grant from the U.S. Public Health Service (GM-08041).Peer reviewe

Comparison of the A-T rich regions and the Bacillus subtilis RNA polymerase binding sites in phage ø29

By using a modification of the BAC spreading method for mounting the DNA for electron microscopy, partial denaturation maps of protein-free ø29 DNA and of ø29 DNA containing protein p3 were obtained. In ø29 P3-DNA1 the protein does not seem to influence the melting of the ends of the molecules. The comparison of the partial denaturation map and the B. subtilis RNA polymerase binding sites indicates that five of the seven early promoters (Al, A2, A3, B2 and C2) are located in A-T rich DNA regions whereas the other two early promoters (Bl and Cl) are located in less A-T rich sites.Peer reviewe

Muscle fructose-1, 6-diphosphatase

Fructose-1, 6-diphosphatase (FDPase), a key enzyme in neoglycogenesis, is generally considered as essentially restricted to liver and kidney although a small activity had been early detected in skeletal muscle (Gomori 1943; Hers and Vanden Berghe 1957). Interest in the regulation of the activity of FDPase has been recently aroused by studies with liver and kidney preparations (Taketa and Pogell 1963; Mendicino and Vasarhely 1963; Krebs 1964 a; Newsholme 1963). This communication reports the occurrence of significant amounts of FDPase in skeletal muscle of frog and rabbit. The frog muscle enzyme has a very great affinity for fructose-1, 6-P (FDP) as substrate and for AMP as allosteric inhibitor. The possible physiological significance of these kinetic properties and of the occurrence of this enzyme in muscle is discussed.This investigation was supported by United States Public Health Service Research Grant GM-08041.Peer reviewe