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Biomedical subjects

J R Greene

Publications and source records attributed to J R Greene.

28 records · Page 2Linked to original sources
PubMed1986

DNA binding by the bacteriophage SPO1-encoded type II DNA-binding protein, transcription factor 1. Site-specific binding requires 5-hydroxymethyluracil-containing DNA.

The bacteriophage SPO1-encoded Type II DNA-binding protein, transcription factor 1 (TF1), forms complexes with specific sites in SPO1 DNA. We have investigated the binding of TF1 to one of its preferred sites in which the normal 5-hydroxymethyluracil (hmUra) of SPO1 DNA has been replaced by thymine and have also investigated the binding of a bacterial Type II DNA-binding protein (from Bacillus stearothermophilus) to the hmUra- and thymine-containing forms of the same DNA segment. Our results show that TF1 binds selectively to this high affinity binding site only in hmUra-containing DNA and that the bacterial Type II DNA-binding protein interacts nonspecifically with both forms of DNA.

Binding Sites↗
PubMed1986

The phage SPO1-specific RNA polymerase, E.gp28, recognizes its cognate promoters in thymine-containing DNA.

The bacteriophage SPO1 gene 28-encoded protein, gp28, directs specific recognition of viral middle promoters in hydroxymethyluracil-containing DNA by the Bacillus subtilis host's RNA polymerase core. Using appropriately sensitive methods of detection, we have shown that discrimination against thymine-containing DNA is not absolute and that the gp28-containing RNA polymerase precisely initiates transcription at two thymine-containing SPO1 middle promoters.

Bacteriophages↗
PubMed1986

Synergistic toxicity of endotoxin and hemoglobin.

Stroma-free hemoglobin (SFH) is advocated as an oxygen-transporting resuscitation solution. Hemoglobin has been shown to enhance endotoxin lethality when given intraperitoneally. It is possible that SFH could interact with endotoxin when used as an oxygen-transporting resuscitation system for trauma victims with contaminating wounds. To assess the effects of these two agents when given intravascularly, rabbits were infused with SFH (1.75 gm/kg) or albumin (controls; 1.75 gm/kg) with and without endotoxin. Two doses of endotoxin were used. At 14.5 ng/kg of Salmonella enteritidis endotoxin, no effect was seen in the albumin group. However, 50% of the hemoglobin group died. At 14.5 micrograms/kg, the albumin group showed hematologic alterations, but all animals lived. All SFH-treated animals died at the higher endotoxin dose. SFH alone caused cardiac abnormalities (bradycardia in 100%, sinus arrhythmias in 30%, and ventricular arrhythmias in 20%), liver abnormalities (necrosis in 40% and 240% increase in alanine aminotransferase activity by 6 hours), and intravascular thrombi (30%). The only hemoglobin-induced abnormality that was more frequent in the presence of endotoxin was ventricular arrhythmias (up to 75% of animals). Thrombin times were approximately 20% larger in all SFH groups compared with the albumin groups. By 6 hours after infusion, endotoxin prolonged the thrombin time even further, despite the lack of fibrinogen consumption. This study shows that endotoxin and SFH exert synergistic toxicity when SFH is given in a clinically relevant dose for an oxygen-transporting resuscitation system. Only minute quantities of endotoxin are needed to produce this phenomenon. We hypothesize that this synergism is endotoxin enhancement of hemoglobin toxicity.

Animals↗
PubMed1986

Toxicity of human hemoglobin solution infused into rabbits.

Aqueous human hemoglobin is being considered as an oxygen-transporting resuscitation solution. However, there are some indications that such solutions may be toxic. We report here that at a clinically significant dose (20% to 30% of the estimated blood volume) of hemoglobin solution (10 gm/dl), toxicity similar to that reported in humans is produced in rabbits. This toxicity is characterized by cardiac rhythm disturbances (25%) and coagulation abnormalities, manifested as intravascular thrombi. Hypoxia, tissue necrosis (brain 29%, liver 25%), and death (22%) were seen. Hypoxia appears to be the cause of death, because the arterial PO2 of rabbits that died was significantly lower (43 +/- 8 mm Hg) than that of rabbits that lived (80 +/- 12 mm Hg). We also report the effect of the method of preparation or degree of purity on the observed toxicity. Stroma-free red blood cell hemolysate, stroma-free hemoglobin, zinc-precipitated hemoglobin, and chromatographically purified hemoglobin were infused. Purification of these solutions ranged from removal of stroma to removal of all detectable nonhemoglobin protein components. In addition, the nonhemoglobin proteins were concentrated and infused into rabbits to compare their effects. All of these hemoglobin solutions induced similar toxicity for rabbits infused with stroma-free hemoglobin. The nonhemoglobin proteins, as well as human serum albumin, exhibited no toxicity. These results demonstrate that, at the clinically relevant dose, hemoglobin or something inherently associated with hemoglobin is toxic.

Animals↗
PubMed1985

Site-specific DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein.

The bacteriophage SP01 genome encodes a virus-specific type II DNA-binding protein, TF1. The bacterial proteins of this ubiquitous and evolutionarily conserved class are thought to bind non-specifically to DNA. In contrast, the experiments described here demonstrate that TF1 binds to specific sites in SP01 DNA. Several of these sites have been characterized by DNase I 'footprinting' and four of them have been shown to overlap strong phage promoters for Bacillus subtilis RNA polymerase holoenzyme. We speculate on the possible structural basis of site-selective DNA binding by a protein of this class.

Bacteriophages↗
PubMed1984

Sequence of the bacteriophage SP01 gene coding for transcription factor 1, a viral homologue of the bacterial type II DNA-binding proteins.

The Bacillus subtilis phage SP01, whose DNA contains 5-hydroxymethyluracil (hmUra) in place of thymine, codes for an abundant, small, basic protein called TF1. TF1 binds preferentially to hydroxymethyluracil-containing DNA and thereby selectively inhibits transcription of such DNA in vitro. The gene for TF1 has been sequenced. We find that this viral protein is a homologue of the ubiquitous bacterial type II DNA-binding proteins. The three-dimensional structure of one of these bacterial proteins has recently been determined. We are able to discern common as well as distinctive features in the amino acid sequence and the three-dimensional structure of the homologous viral protein.

Amino Acid Sequence↗
PubMed1982

SP01 gene 27 is required for viral late transcription.

The SP01 mutant sus HA20 (gene 27) was found to be defective for synthesis of viral late RNA. It is known that gene 27 is also required for viral DNA replication. The SP01 gene 27 product resembles the T4 gene 45 product, which also has a dual role in viral DNA replication and late transcription.

Bacillus subtilis↗
PubMed1981

Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus N and NS proteins.

The complete nucleotide sequences of the vesicular stomatitis virus (VSV) mRNA's encoding the N and NS proteins have been determined from the sequences of cDNA clones. The mRNA encoding the N protein is 1,326 nucleotides long, excluding polyadenylic acid. It contains an open reading frame for translation which extends from the 5'-proximal AUG codon to encode a protein of 422 amino acids. The N and mRNA is known to contain a major ribosome binding site at the 5'-proximal AUG codon and two other minor ribosome binding sites. These secondary sites have been located unambiguously at the second and third AUG codons in the N mRNA sequence. Translational initiation at these sites, if it in fact occurs, would result in synthesis of two small proteins in a second reading frame. The VSV and mrna encoding the NS protein is 815 nucleotides long, excluding polyadenylic acid, and encodes a protein of 222 amino acids. The predicted molecular weight of the NS protein (25,110) is approximately one-half of that predicted from the mobility of NS protein on sodium dodecyl sulfate-polyacrylamide gels. Deficiency of sodium dodecyl sulfate binding to a large negatively charged domain in the NS protein could explain this anomalous electrophoretic mobility.

Amino Acid Sequence↗