Fractionation of deoxyribonucleic acid from phage-infected bacteria

1. DNA has been isolated in 90% yield from T5-infected cultures of Escherichia coli ;pulse'-labelled with [(3)H]thymidine. It had a buoyant density in caesium chloride solution identical with the DNA of mature T5 phage, and no components of unusual buoyant density were detected. 2. The DNA preparation was resolved into two major components of differing specific activity on a column of kieselguhr coated with methylated serum albumin. The DNA of high specific activity could be eluted from the column only with 2n-ammonia, and the firm binding did not appear to be due to an artifact of preparation. 3. A similar fractionation into two DNA components of differing specific activity was observed when the ;pulse'-labelled culture was lysed with sodium dodecyl sulphate and the lysate rocked with phenol. The DNA of high specific activity was found in the interface precipitate between the phenol and aqueous layers. 4. The amounts of DNA in the two fractions were measured at different times after infection and the radioactivity content of each was determined at various times after a short ;pulse' of [(3)H]thymidine. The interface fraction contained the replicating phage DNA, and the DNA from mature phage particles appeared in the aqueous fraction. 5. Analogous results were obtained with T2-infected E. coli. In the presence of chloramphenicol the DNA in the interface fraction was not converted into DNA extractable into the aqueous layer. Since chloramphenicol prevents the condensation of DNA into phage heads, it is suggested that any DNA in extended configuration is trapped inside the rigid-layer framework of the cell wall. 6. Treatment with lysozyme released much of the DNA from the interface precipitate. This DNA was firmly bound by the chromatographic column and had the same buoyant density in caesium chloride solution as normal T5-phage DNA. Sucrose-gradient sedimentation studies showed that it was heterogeneous and that as much as 60% sedimented faster than T5-phage DNA.     

The enzymic estimation of glutamate and glutamine

A method of estimating glutamic acid is described, based on its dehydrogenation by glutamate dehydrogenase coupled, by means of N-methylphenazine methosulphate, to the reduction of tetrazolium salts. The method is suitable for the estimation of 0-0.3mumole of glutamic acid. The response is linear, but not stoicheiometric: possible reasons for this are discussed. If suitable precautions are taken, the use of a partially purified preparation of glutaminase makes it possible to estimate glutamine also.     

The relation of 3-deoxy-2-oxo-octonate to the serological and physical properties of a lipopolysaccharide from a rough strain of Escherichia coli

1. A water-soluble preparation of lipopolysaccharide was isolated from the extracellular lipopolysaccharide-phospholipid-protein complex formed by Escherichia coli A.T.C.C. 12408. 2. Heating at 100 degrees and pH4.6 caused a rapid decrease in serological activity concomitant with the release of 3-deoxy-2-oxo-octonate; aggregation of the molecules also occurred. 3. Further evidence that the release of 3-deoxy-2-oxo-octonate is related to loss of serological activity was obtained by comparing the effects of heating for 1hr. at 100 degrees and pH values between 5.0 and 9.0; detectable changes still occurred at pH7.5. 4. 3-Deoxy-2-oxo-octonate was isolated from the products of hydrolysis of lipopolysaccharide and shown to be an effective inhibitor of the precipitin reaction between lipopolysaccharide and homologous antiserum. 5. The possibility that 3-deoxy-2-oxo-octonate is joined to the lipopolysaccharide through a phosphodiester linkage is discussed.     

Effects of prolonged incubation of isolated fat cells on their response to hormones stimulating lipolysis and glucose metabolism

1. The metabolism of isolated fat cells from parametrial adipose tissue of starved normal rats was studied during 8hr. incubation. 2. There was a three- to eight-fold increase in conversion of glucose into carbon dioxide, fatty acids and glycerol during the fourth to eighth hours of incubation in 4% albumin buffer over that seen during the first 4hr. of incubation. 3. The addition of growth hormone and dexamethasone to fat cells at the start of the incubation period accelerated lipolysis during the first 4hr. of incubation but no further effect was seen during the fourth to eighth hours of incubation. Addition of growth hormone and dexamethasone to fat cells that had been incubated for 4hr. did not accelerate lipolysis during the next 4hr. whether fat cells were incubated with or without glucose. 4. Fat cells incubated for prolonged periods also displayed a reduced sensitivity to the lipolytic action of adrenocorticotrophic hormone. 5. During prolonged incubation there was no damage to the cells as judged by the retention of two soluble cytoplasmic enzymes, lactate dehydrogenase and malate dehydrogenase, within the cells.