Site-specific properties of Tn7 transposition into the E. coli Chromosome

Summary A study has been made of the insertional properties of transposon Tn7, a 14 kilobase transposable element encoding resistances to trimethoprim, streptomycin and specitinomycin. It has previously been shown that Tn7 transposes at a low frequency and with low specificity into multiple sites in large transmissible plasmids. However, Tn7 transposes with extrame specificity and at high efficiency into the E. coli chromosome. In all cases we have studied, insertion of Tn7 into the chromosome has occurred at a unique site and with a unique orientation. A combination of genetic and biochemical techniques have been used to precisely locate this site on the E. coli chromosome to minute 82 on the linkage map between markers glmS and uncA.To investigate the nature of this highly specific transpositional event, a small region of the E. coli chromosome that includes the unique site, was cloned into the plasmid vector pBR322. Subsequently a lkb restriction fragment, including the Tn7 insertion site, was sub-cloned from this plasmid into the plasmid pACYC184. We show that Tn7 transposes into both these plasmid recombinants with the frequency and specificity characteristie of the E. coli chromosome.     

Deoxyribonucleic acid relatedness inYersinia enterocolitica andYersinia enterocolitica-like organisms

Yersinia enterocolitica andY. enterocolitica-like strains were characterized by DNA relatedness. These strains formed four distinct DNA relatedness groups: (i) the 5 classical biotypes ofY. enterocolitica sensu stricto as designated by Wauters; (ii) strains that are rhamnose positive and also positive in tests for melibiose, α-methyl-d-glucoside, raffinose, and Simmons' citrate; (iii) strains that are rhamnose positive but negative in tests for melibiose, α-methyl-d-glucoside, and raffinose; (iv) sucrose-negative, Voges-Proskauer-negative, trehalose-positive strains.     

Laser microirradiation of kinetochores in mitotic PtK2 cells

Irradiation of the kinetochore region of PtK₂ chromosomes by laser light of 532 nm was used to study the function of the kinetochore region in chromosome movement and to create artificial micronuclei in cells. When the sister kinetochores of a chromosome were irradiated at prometaphase, the affected chromosome detached from the spindle and exhibited no further directed movements for the duration of mitosis. The chromatids of the chromosome remained attached to one another until anaphase, at which point they separated. No poleward movement of the chromatids was observed, and at telophase they passively moved to one of the daughter cells and were enclosed in a micronucleus. The daughter cell containing the micronucleus was then isolated by micromanipulation and followed through subsequent mitoses. At the next mitosis, two chromosomes, each with two chromatids, condensed in the micronucleus. These chromosomes did not attach to the spindle and showed chromatid separation, but no poleward movements at anaphase. They were again enclosed in micronuclei at telophase. The third generation mitosis was similar to the second. Occasionally, both the irradiation-produced and naturally occurring micronuclei exhibited no chromosome condensation at mitosis. Feulgenstained monolayers of PtK₂ cells with naturally occurring micronuclei showed that some micronuclei stain positive for DNA and others do not. This finding raises questions about the fate of chromosomes in a micronucleus.     

The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization

Substoichiometric concentrations of cytochalasin D inhibited the rate of polymerization of actin in 0.5 mM MgCl2, increased its critical concentration and lowered its steady state viscosity. Stoichiometric concentrations of cytochalasin D in 0.5 mM MgCl2 and even substoichiometric concentrations of cytochalasin D in 30 mM KCl, however, accelerated the rate of actin polymerization, although still lowering the final steady state viscosity. Cytochalasin B, at all concentrations in 0.5 mM MgCl2 or in 30 mM KCl, accelerated the rate of polymerization and lowered the final steady state viscosity. In 0.5 mM MgCl2, cytochalasin D uncoupled the actin ATPase activity from actin polymerization, increasing the ATPase rate by at least 20 times while inhibiting polymerization. Cytochalasin B had a very much lower stimulating effect. Neither cytochalasin D nor B affected the actin ATPase activity in 30 mM KCl. The properties of cytochalasin E were intermediate between those of cytochalasin D and B. Cytochalasin D also stimulated the ATPase activity of monomeric actin in the absence of MgCl2 and KCl and, to a much greater extent, stimulated the ATPase activity of monomeric actin below its critical concentration in 0.5 mM MgCl2. Both above and below its critical concentration and in the presence and absence of cytochalasin D, the initial rate of actin ATPase activity, when little or no polymerization had occurred, was directly proportional to the actin concentration and, therefore, apparently was independent of actin-actin interactions. To rationalize all these data, a working model has been proposed in which the first step of actin polymerization is the conversion of monomeric actin-bound ATP, A . ATP, to monomeric actin-bound ADP and Pi, A* . ADP . Pi, which, like the preferred growing end of an actin filament, can bind cytochalasins.     

Determination of serum protein concentration in nanoliter blood samples using fluorescamine or 9-phthalaldehyde.

Fluorometric procedures were developed to permit measurement of total protein concentration in nanoliter serum samples, using either fluorescamine or o-phthalaldehyde. The sensitivities of assays using these two reagents were similar, but the o-phthalaldehyde method was found to be somewhat simpler and more reproducible. Accurate measurements could be obtained on serum samples of 4 to 5 nl with either reagent, by using a serum standard. Fluorescence differed considerably among individual serum proteins, albumin generally showing greater fluorescence than globulins. Small molecular weight species in serum did not contribute appreciably to total serum fluorescence with either reagent.     

Comparison of human and bovine protoporphyria.

Protoporphyria (PP) is an inherited disorder of porphyrin metabolism in man in which there is excessive accumulation and excretion of protoporphyrin. Recently, a similar disorder has been described in cattle. In this report, the clinical, biochemical, and genetic features of bovine and human PP are compared. Human and bovine PP are characterized by photosensitivity and elevation of erythrocyte and fecal protoporphyrin levels. In both disorders, a deficiency of heme synthase activity is present in all tissues which have been examined. The diseases differ clinically in that hepatobiliary disease has been found thus far only in human PP. They also have different inheritance patterns. Human PP is an autosomal dominant disease, while initial studies strongly suggest that there is an autosomal recessive pattern of inheritance in bovine PP.     

Effect of different carbon sources on the biosynthesis of polyunsaturated fatty acids ofα-linolenic acid family in culture of minimal deviation hepatoma 7288 c cells

Summary The effect of three different carbon sources on the biosynthesis of polyunsaturated fatty acids of the-linolenic acid series was investigated in hepatoma tissue culture (HTC) cells. Alpha linolenic acid was converted to higher homologs by a desaturating route that synthetized mainly 18:4 (6, 9, 12, 15), 20:4 (8, 11, 14, 17) and 20:5 (5, 8, 11, 14, 17) and an elongating route that produced 20:3 (11, 14, 17) and 20:4 (5, 11, 14, 17) acids. Fasting decreased both biosynthetic routes whereas glucose reactivated only the elongating pathway. Lactalbumin hydrolysate enhanced significantly only the desaturating route whereas glycerol was inactive. Glucose and aminoacids increased similarly the incorporation of labeled linolenic acid in the cells. The results are independent of hormonal effects.Members of the Carrera del Investigador Científico of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

DNA relatedness among species of Enterobacter and Serratia.

Species of Enterobacter and Serratia were examined for deoxyribonucleic acid relatedness to Klebsielleae, to atypical erwiniae, and to other members of Enterobacteriaceae. Deoxyribonucleic acid hybridization and then hydroxyapatite chromatography was the technique used to assess relatedness. Strains of Enterobacter cloacae formed two separate hybridization groups that correlate with the presence or absence of yellow pigment. Pigmented E. cloacae were 75-100% related, but they were only 40-50% related to unpigmented strains. Conversely, unpigmented strains were 70% or more related but were only 40-50% related to the pigmented strains. Both pigmented and unpigmented E. cloacae were 40-45% related to Enterobacter aerogenes and klebsiellae, and 20-30% related to Serratia species and Enterobacter hafniae. Atypical erwiniae were highly related to E. cloacae. Serratia marcescens strains formed one closely related group. Serratia liquefaciens strains formed a single, more disperse, relatedness group, as did isolates of Serratia rubidaea. These species were related throughout a substantial portion of their genomes. A group of lysine-positive "Citrobacter-like" strains were 40-50% related to Serratia species. Only four E. hafniae strains were tested. Two of these were highly related, while the other two were only 50% related to the reference strain. Enterobacter hafniae was only 15-20% related to other Enterobacteriaceae.     

Effect of epinephrine on the oxidative desaturation of fatty acids in the rat.

The effect of epinephrine on the oxidative desaturation of fatty acids by liver microsomal preparations of rats has been studied. Administration of epinephrine (1 mg/kg body weight) produced a significant decrease in desaturation of [l-14C]=linoleic acid to gamma-linolenic acid and of [L-14C]alpha-linolenic acid to actadeca-6,9,12,15-tetraenoic acid 12 hr after the infection. Lower doses produced a lesser effect on the delta6-desaturation activity. Epinephrine administration modified the V max of linoleic acid desaturation but not the K m. There was also a slight increase in palmityl desaturation activity. The effect of epinephrine on delta6-desaturation activity was postulated to be mediated through an enhancement of the intracellular cyclic AMP levels that lead to an increase of a glucose metabolite. This metabolite would inhibit delta6-desaturation activity.