Localization of two human homologs, HHR6A and HHR6B, of the yeast DNA repair gene RAD6 to chromosomes Xq24-q25 and 5q23-q31.

The chromosomal localizations of two closely related human DNA repair genes, HHR6A and HHR6B, were determined by in situ hybridization with biotinylated probes. HHR6A and HHR6B (human homolog of yeast RAD6) encode ubiquitin-conjugating enzymes (E2 enzymes), likely to be involved in postreplication repair and induced mutagenesis. The HHR6B gene was assigned to human chromosome 5q23-q31, whereas the HHR6A gene was localized on the human X chromosome (Xq24-q25). This latter assignment was confirmed with an X-specific human-mouse/hamster somatic cell hybrid panel. Southern blot analysis points to an X and an autosomal localization of HHR6A and HHR6B, respectively, in the mouse. The potential involvement of these genes in human genetic disorders is discussed.     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage phiR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10 transconjugants per g of soil when 10 donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx RpP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Characterization of caulobacters isolated from wastewater treatment systems.

Caulobacters are generally assumed to be found only in environments of low organic content; however, we readily isolated strains from a variety of sewage treatment system designs and locations, and 33 distinct strains were characterized. Most were morphologically similar, having the crescent-shaped cell body, short stalk, and hexagonally packed, paracrystalline surface (S) layer characteristic of several Caulobacter crescentus laboratory strains. Upon closer examination, they were distinguishable on the basis of protein band profiles on polyacrylamide gel electrophoresis, gross colony characteristics, or holdfast composition or by DNA restriction fragment length polymorphism analysis with flagellin and S-layer gene probes. Most of the isolates contained one or more high-molecular-weight plasmids and were resistant to a number of antibiotics, characteristics generally not shared with caulobacters isolated from other sources. Six of the 33 strains were retained because they did not fit the typical isolate profile; these strains are overrepresented in our collection compared with their relative proportion in wastewater treatment systems. By colony hybridization and restriction fragment length polymorphism analysis, all of these and one typical isolate showed less homology than the others to the surface array gene of a laboratory strain (C. crescentus CB15), and three hybridized less strongly with the flagellin gene from the same strain. In sum, although the strains were distinguishable, caulobacters from the wastewater treatment systems we examined were relatively homogenous, were similar to characterized laboratory strains, and, with exceptions, could probably be reliably detected as a group by gene probes derived from C. crescentus strains.     

Measurement and effects of gel matric potential and expressibility on production of morphogenic callus by cultured sugarbeet leaf discs

During studies to optimize production of morphogenic callus from cultured leaf discs of sugarbeet (Beta vulgaris L.) large differences were observed associated with the gelling agent employed. Water availability, as determined mainly by gel matric potential, was found to be the dominant factor. A simple method was devised to measure the relative matric potential of different gels. A precisely moistened filter-paper disc was placed on the gel surface, allowed to equilibrate, removed and weighed. The relative gain or loss of water from the paper disc was a measure of the matric potential of the gel and varied with both gel type and concentration. Leaf disc expansion and production of callus-derived embryos and shoots were shown to be directly proportional to gel matric potential. Water availability may also be affected by the ease with which liquid is expressed from gels in response to localized pressure caused by explant expansion and contortion. This property, called gel expressibility, was easily measured with a weight and capillary pipette and shown also to vary with gel type and concentration. Validity of the technique for measuring relative matric potential was verified physiologically by culturing leaf discs on filter-paper overlays to eliminate expressibility differences among gels. Additionally, comparison of leaf disc growth on uncovered gel surfaces versus filter-paper overlays demonstrated the contribution of liquid expression to overall water availability. Expression of liquid by explants on uncovered gel surfaces greatly enhanced the production of morphogenic callus.     

Identification and characterisation of an Acinetobacter sp. capable of assimilation of a range of cyano-metal complexes,free cyanide ions and simple organic nitriles

Summary An Acinetobacter sp. strain RFB1 was shown to be capable of degrading a wide range of cyano-metal complexes, simple cyanide salts and simple organic nitrile compounds. The enzymatic activity responsible for this degradation was located in an extracellular lipid complex. This complex could not be resolved into the constitutive components under standard conditions without loss of activity. Offsprint requests to: I. Finnegan     

Localization and quantification of extracellular polymers in methanogenic granular sludge

Summary Extracellular polymers were localized and quantitatively analysed in methanogenic granular sludge cultivated on either propionate or ethanol in laboratory upflow anaerobic sludge-blanket (UASB) reactors. Electron microscopical analysis of ultrathin sections of the two sludge types stained with ruthenium red revealed the presence of extracellular polymers with different densities and structures. For quantification, granular sludge from a large-scale UASB reactor at a liquid sugar plant was also included in this study. A three-step physical disintegration procedure was used to extract water-soluble extracellular material from the granules. After each disintegration step the extracts were analysed for polysaccharides and proteins. Cell damage and thus the contribution of intracellular proteins and polysaccharides was estimated simultaneously by the determination of free DNA and free ATP in the extracts. After two extraction steps, up to 3.5 mg polysaccharides/g organic material and 5.5 mg protein/g organic material were extracted, whereas no significant increase in DNA was detected. The role of extracellular polymers in granular stability is discussed. Offsprint requests to: A. J. B. Zehnder     

Lymphocyte activation and serine-esterase induction following recombinant interleukin-2 infusion for lymphomas and acute leukaemias

Summary C57BL mice inoculated with radiation leukemia virus (RadLV) develop preleukemic cells long before the onset of leukemia. These cells are potentially immunogenic but fail to elicit an immune response in the host because of the appearance of virus-specific suppressor T cells. We have studied the effect of polysaccharide K (PSK) on the generation of RadLV-specific cell-mediated immune responses in vitro. Long-term exposure to PSK in culture potentiated the ability of immunized T cells to respond to a RadLV-induced lymphoma. It also abrogated the suppressive activity of suppressor T cells and simultaneously boosted the ability of reactive T cells to respond. The dual immunostimulating activity of PSK resulted in the generation of T cytotoxic lymphocytes that could lyse lymphoma cells in vitro. The results suggest that PSK could be used as a prophylactic immune response modifier in preleukemia.     

A highly repeated DNA sequence in Arabidopsis thaliana

Summary Three members of a family of highly repeated DNA sequences from Arabidopsis thaliana have been cloned and characterized. The repeat unit has an average length of 180 bp and is tandemly repeated in arrays longer than 50 kb. This family represents more than one percent of the Arabidopsis genome. Sequence comparisons with tandemly repeated DNA sequences from other Cruciferae species show several regions of homology and a similar length of the repeat unit. Homologies are also found to highly repeated sequences from other plant species. When the sequence CCGG occurs in the repeated DNA, the inner cytosine is generally methylated.     

Isolation and Characterization of Pyrophosphate:D-Fructose-6-phosphate 1-Phosphotransferase from Cucumber Seeds

Pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase waspurified over 700-fold from germinating cucumber (Cucumis sativuscv. Fletcher) seeds. The purified enzyme has a specific activityof 5.2 µmol.min^–1.mg protein^–1 in the presenceof 1 µM fru-2,6-P₂. The pH optima is similar for boththe forward and reverse reactions (pH 7.5–7.8). Magnesium,manganese and cobalt activate the enzyme, with the highest affinitybeing for magnesium. The enzyme exhibits normal Michaelis-Mentenkinetics in both the presence and absence of fru-2,6-P₂. Half-maximumactivation of the enzyme was obtained with 35 nM fru-2,6-P2.Fru-2,6-P₂ stimulates activity by increasing V_max and increasingthe affinity for fru-6-P, fru-1,6-P₂ and PPi. Phosphate causesnoncompetitive inhibition with respect to both fru-6-P and PPi.On the basis of the steadystate substrate interaction and Piinhibition data a sequential ternary complex mechanism is proposed. (Received April 28, 1986; Accepted July 9, 1986)     

A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b

We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press, the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by (1) selective solubilization with sodium dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents and heat; and (3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required.