Plasmids useable as gene-cloning vectors in an in vitro packaging by coliphage λ: “cosmids”

A plasmid which contains a cos site of λ and can be packaged into lambda bacteriophage particles is termed a “cosmid”. Such plasmids can be used as gene cloning vectors in conjunction with an in vitro packaging system. The properties of a new series of cosmids based on the ColE1 replicon are described, including small temperature-sensitive plasmids which have lost mobilisation functions and carry no IS sequences. Amongst these plasmids are vectors for XmaI, BglII, BamHI, HindIII, PstI, KpnI, SalI and EcoRI. It is demonstrated that by using cosmids in particular size ranges these plasmids provide a high efficiency cloning system which yields essentially only hybrid clones without resort to a second selection or screening step, and without prior modification (e.g. phosphatase) treatment of the DNA.Attempts were made to optimise the cloning properties of the cosmid system. An Escherichia coli “gene bank” was obtained with an efficiency of 5·10⁵ clones per μg of E. coli DNA, and in which any particular unselected marker may be found in about one out of every 400 clones.It was demonstrated that deletion of mobilisation functions leads to loss of ability to form relaxation-complex without affecting copy number or segregation properties of the temperature-sensitive derivatives. The vectors are amplifiable in chloramphenicol to make up about 50% of the total cellular DNA.     

Isomerization of 1,3,4,5,6-Pentachlorocyclohexene-1 Isomers in Dimethyl Sulfoxide

In dimethyl sulfoxide, isomerization of 1,3,4,5,6-pentachlorocyclohexene-1 isomers was studied. Exchange of the allylic chlorine was indicated by the experiments with ³⁶Cl-labeled compounds. Isomerization at the position-3 proceeded faster than at the position-6. Initial reaction rates for the isomers decreased in the order: 34/56 > 35/46 (δ) > 346/5> 356/4 ? 36/45 (γ)^??. The specific rates at the initial stages increased by increasing the initial concentration of the substrates. After a prolonged reaction, the γ-isomer (36/45) was the most abundant component.

The isomers of the configuration: (34/56), (346/5), and (356/4) were isolated for the first time, on which PMR studies were performed.     

Mannose-specific adherence of Escherichia coli to BHK cells that differ in their glycosylation patterns

Abstract We measured the mannose-specific adherence of radiolabeled Escherichia coli , carrying type 1 fimbriae, to monolayers of wild-type baby hamster kidney (BHK) cells and to 3 ricin-resistant mutants defective in the synthesis of complex N -linked oligosaccharide units. Ric^R14, a mutant accumulating N-linked oligomannose units in its glycoproteins at the expense of complex ( N -acetyllactosamine) units, bound the largest number of bacteria, about 4 times more than the wild-type cells. The mutant cells in suspension were also readily agglutinated by the bacteria, while no agglutination of wild-type cells occurred under the conditions used. Ric^R21, a mutant which accumulates hybrid structures, bound about twice as many bacteria as wild-type cells, and was agglutinated by the bacteria to a lesser extent than Ric^R14. Binding and agglutination of Ric^R19, also presumed to accumulate hybrid structures, were the same as those of Ric^R14. These results provide evidence that oligomannose and hybrid units of cell surface glycoproteins serve as preferred receptors for mannose-specific E. coli. Lectin-resistant mutants are therefore useful for the investigation of sugar-specific adherence.     

The relationship of biomass,polysaccharide and H2 formation in the wild-type and nifA/nifB mutants of Rhodobacter capsulatus

The production of biomass, polysaccharide storage material and H₂ from malate was studied in the wild-type and mutants RdcI, RdcII and RdcI/cII of Rhodobacter capsulatus. The mutants are defective in either copy I, copy II or both copies of the nitrogenase genes nifA and nifB. Stationary phase levels of biomass, polysaccharide and H₂ were determined in phototrophic batch cultures grown with 30 mM of d,l-malate and either 2, 5, or 8 mM of ammonium or 7 mM of glutamate. Calculation of the amounts of malate converted into the three products revealed that, at 8 mM of ammonium and 7 mM of glutamate, malate consumption and product formation were balanced. But with decreasing ammonium concentrations malate not converted into biomass was utilized with decreasing efficiency in polysaccharide and H₂ formation. This suggests formation of unknown products at the lower ammonium concentrations. Under conditions of optimal N supply, 80% of the malate not used for biomass production was converted by the wild-type and strain RdcII to H₂ and CO₂. Mutant RdcI exhibited slightly decreased H₂ production. The double mutant did not evolve H₂ but accumulated increased amounts of polysaccharide. However, the amounts of polysaccharide were lower than should be expected if all of the spare malate, not utilized by the double mutant for H₂ production, was converted into storage material. This and incomplete conversion of malate into known products at low ammonium supplies suggests that polysaccharide accumulation does not compete with the process of H₂ formation for malate.     

Synthesis and degradation of alcohol dehydrogenase in wild-type and Adh-null activity mutants of Drosophila melanogaster

Both the amount and the size of alcohol dehydrogenase-like cross-reacting material was determined in 14 ethyl methanesulfonate (EMS)-induced alcohol dehydrogenase-null activity mutants. In 11 mutants cross-reacting material of the same apparent molecular weight as alcohol dehydrogenase was detected, while in 3 mutants no cross-reacting material was found. In all cases, the amount of cross-reacting material found in the mutants was lower than that in wild-type flies. High, intermediate, and low cross-reacting material-producing mutants showed similar initial rates of incorporation of labeled amino acid into alcohol dehydrogenase-like protein, presumably reflecting similar rates of synthesis. If the rate of synthesis of cross-reacting material is the same in the mutants as in the wild type, then the different levels of cross-reacting material must be due to different rates of degradation.Supported by NIH Grants GM-18254 and ES-01527 and DOE Contract EY-76-S-2965.     

A possible physiological function of the oxygen-photoreducing system of Rhodospirillum rubrum

Anaerobic suspensions of Rhodospirillum rubrum cells which had been grown in the dark under low oxygen tension showed only a small increase of their ATP content when illuminated for 30 s. The same suspensions failed to start immediate growth in the light. Both high light-induced ATP levels and immediate phototrophic growth were elicited by small amounts of oxygen which were insufficient by themselves to raise the ATP levels or to support growth in the dark. The oxygen requirement for growth disappeared after some time of anaerobic illumination and was not observed in suspensions of cells which had been grown in the light under anaerobiosis. Furthermore, these phototrophic cells reached the maximum levels of ATP when illuminated in the absence of oxygen.Strain F11, a mutant derivative of Rhodospirillum rubrum which lacked the ability to photoreduce oxygen in vitro, needed abnormally high amounts of oxygen to increase its ATP levels and to grow in the light. Besides, KCN inhibited the increase of ATP levels in illuminated mutant cells but not wild type cells. An additional difference between both strains was that the oxygen requirement for growth did not disappear in the mutant after some time of anaerobic incubation in the light.To explain these observations, it is proposed that the photosynthetic system of semiaerobically-grown Rhodospirillum rubrum becomes overreduced under anaerobiosis. The oxygen-photoreducing system, which is impaired in the mutant, is apparently used to oxidize the photosynthetic system to its optimal redox state, carrying electrons to oxygen or to other endogenous acceptors which are formed during incubation in the light. The mutant seems to replace the defective system by a cyanide-sensitive pathway which may reduce oxygen but not the alternative endogenous acceptors.     

Biochemical analysis of galactose induced bacteriostasis ingalT mutants ofescherichia coli K 12

Escherichia coli mutants completely defective in galactose-1-phosphate uridyl transferase (EC 2.7.7.10) and growing in glycerol medium undergo rapid cessation of growth when exposed to galactose. Toxicity due to galactose is equally pronounced when glycerol is replaced by other carbon sources, like succinate and proline. Gas chromatographic analysis failed to detect even trace amounts of galactitol. Moreover, galactose-1-phosphate had no inhibitory role on some of the critical enzymes of cellular metabolism. General loss of energy (ATP) due to futile phosphorylation of galactose is probably the cause of bacteriostasis. ThegalT mutants can serve as models of human transferaseless galactosemia only to a limited extent     

Sensitivity of Escherichia coli and Pseudomonas aeruginosa to avoparcin, flavomycin and virginiamycin

Abstract The sensitivities of wild-type, rough, deep-rough and antibiotic-hypersensitive strains of Escherichia coli and Pseudomonas aeruginosa to avoparcin, flavomycin and virginiamycin are described. Avoparcin was least active against these strains. Overall, the results suggested that resistance of wild-type cells to these antibiotics resulted from their exclusion from the cells.