A dnaB-like protein of Pseudomonas aeruginosa.

A dnaB-like protein from P. aeruginosa was purified to near homogeneity using as an assay the immunoprecipitation by E. coli dnaB antiserum in a solid-phase. In the chromatographic characteristics including the affinity to immobilized ATP the dnaB-like protein of P. aeruginosa is similar to the dnaB protein of E. coli with the exception that it does not bind to heparin-Sepharose. The dnaB-like protein has a native molecular weight of about 320,000 as determined by glycerol gradient sedimentation. It consists of several identical subunits of molecular weight of 56,000 as measured in a denaturing SDS gel. Associated with the enzyme is a DNA-dependent ATPase- and helicase activity. The dnaB-like protein is similar to the E. coli dnaB protein with regard to the binding of ATP gamma S and the formation of a ternary complex consisting of the enzyme, ATP gamma S, and phi X174 DNA. However, the enzyme of P. aeruginosa is inactive in a phi X174 DNA-dependent in vitro dnaB complementation assay using an E. coli dnaBts extract.     

Surface areas,lengths and volumes of Picea abies (L.) Karst. needles: determination,biological variability and effect of environmental factors

Summary A method for the rapid determination of the lengths and surface areas of very large samples of needles of Picea abies (L.) Karst. using a computer-aided image analysis system was developed. Two independent methods for measuring non-destructively the volumes of individual needles and of all needles attached to a twig were devised. The surface areas and lengths of about 38000 needles sampled from the three youngest needle age-classes (1986, 1985, 1984) of 48 trees approximately 130 years old at four sites in the Fichtelgebirge mountains (N. E. Bavaria, FRG) were measured. The frequency distributions of lengths and areas for each site and age-class are given. Variability of needle size was fairly large. Even though the sites differed in climate, soil, and air pollution levels no consistent effect of these factors on needle size could be detected. Needle lengths and surface areas did not correlate with either the total chlorophyll content of the needles or the degree of crown thinning. The needle surface area (in mm²) of fully developed P. abies needles can be estimated by the empirical equation surface area = 4.440 x needle length -24.8 (r = 0.937), and the needle volume (in mm³) by needle volume = 0.208 x projected needle area ^1.353 (r = 0.969).     

Molecular analysis and pathogenesis of the feline aplastic anemia retrovirus, feline leukemia virus C-Sarma.

We describe the molecular cloning of an anemogenic feline leukemia virus (FeLV), FeLV-C-Sarma, from the productively infected human rhabdomyosarcoma cell line RD(FeLV-C-S). Molecularly cloned FeLV-C-S proviral DNA yielded infectious virus (mcFeLV-C-S) after transfection of mammalian cells, and virus interference studies using transfection-derived virus demonstrated that our clone encodes FeLV belonging to the C subgroup. mcFeLV-C-S did not induce viremia in eight 8-week-old outbred specific-pathogen-free (SPF) cats. It did, however, induce viremia and a rapid, fatal aplastic anemia due to profound suppression of erythroid stem cell growth in 9 of 10 inoculated newborn, SPF cats within 3 to 8 weeks (21 to 58 days) postinoculation. Thus, the genome of mcFeLV-C-S encodes the determinants responsible for the genetically dominant induction of irreversible erythroid aplasia in outbred cats. A potential clue to the pathogenic determinants of this virus comes from previous work indicating that all FeLV isolates belonging to the C subgroup, an envelop-gene-determined property, and only those belonging to the C subgroup, are potent, consistent inducers of aplastic anemia in cats. To approach the molecular mechanism underlying the induction of this disease, we first determined the nucleotide sequence of the envelope genes and 3' long terminal repeat of FeLV-C-S and compared it with that of FeLV-B-Gardner-Arnstein (mcFeLV-B-GA), a subgroup-B feline leukemia virus that consistently induces a different disease, myelodysplastic anemia, in neonatal SPF cats. Our analysis revealed that the p15E genes and long terminal repeats of the two FeLV strains are highly homologous, whereas there are major differences in the gp70 proteins, including five regions of significant amino acid differences and apparent sequence substitution. Some of these changes are also reflected in predicted glycosylation sites; the gp70 protein of FeLV-B-GA has 11 potential glycosylation sites, only 8 of which are present in FeLV-C-S.     

Reactions in vitro of the DNA polymerase-primase from Xenopus laevis eggs. A role for ATP in chain elongation

A form of DNA polymerase alpha was purified several thousandfold from a protein extract of Xenopus laevis eggs. The enzyme effectively converts, in the presence of ribonucleoside triphosphates, a circular single-stranded phage fd DNA template into a double-stranded DNA form and, therefore, must be associated with a DNA primase. We first show by gel electrophoresis in the presence of sodium dodecyl sulfate that both enzymatic activities, DNA polymerase and primase, most probably reside on a greater than 100 000-Da subunit of the DNA polymerase holoenzyme. We then assayed the polymerase-primase at various template/enzyme ratios and found that the DNA complementary strand sections synthesized in vitro belong to defined size classes in the range of 600-2000 nucleotides, suggesting preferred start and/or stop sites on the fd DNA template strand. We show that the stop sites coincide with stable hairpin structures in fd DNA. We have used a fd DNA template, primed by a restriction fragment of known size, to show that the polymerase-primase stops at the first stable hairpin structure upstream from the 3'-OH primer site when the reaction was carried out at 0.1 mM ATP. However, at 2 mM ATP the enzyme was able to travers this and other stop sites on the fd DNA template strand leading to the synthesis of 2-4 times longer DNA strands. Our results suggest a role for ATP in the polymerase-primase-catalyzed chain-elongation reaction.     

Pectinases in Aqueous Extracts of Ditylenchus dipsaci

Aqueous extracts of a population of Ditylenchus dipsaci isolated from onion and maintained monoxenically on onion callus contained endo-polygalacturonase (endo-PG) and endo-pectinmethyltranseliminase (endo-PMTE). In viscometric tests pH 4.2 and 4.0 were optimal for degradation of sodium polypectate and pectin N.F., respectively, by endo-PG. Endo-PMTE reduced viscosity of pectin N.F. optimally at pH 8.5 or above. Activity was dependent on CaCl₂. Pectinmethylesterase activity was not detected in water, NaCl, or sucrose extracts of these nematodes. The extracts macerated potato tuber tissue, onion cotyledonary tissue, and strips of onion epidermis from the ventral surface of onion bulb scales at pH 4.2, 5.3, and 6.2. Pectin could not be localized with hydroxylamine-ferric chloride reagent in macerated tissues treated for 24 hr with active extract.     

A Comparison of Pectinases from Ditylenchus dipsaci and Allium cepa Callus Tissue

Ground and whole Ditylenchus dipsaci maintained on onion callus contain no culturable micro-organisms when tested with five check media. Healthy onion callus does not produce pectolytic enzymes. Pectolytic enzymes are present in infected callus. These enzymes are, however, associated with resident nematodes and not host tissues. These results suggest that D. dipsaci is the actual source of the endo-polygalacturonase and endo-pectinmethyltrans-eliminase extracted from them.     

THE SEPARATION OF DIFFERENT CELL CLASSES FROM LYMPHOID ORGANS : III. The Purification of Erythroid Cells by pH-Induced Density Changes

1. Mammalian erythrocytes swell as the pH of the isotonic suspending medium is lowered, as a direct consequence of the specialized permeability properties of the erythrocyte membrane. Lymphocytes and granulocytes from a variety of sources did not exhibit this property. 2. The behaviour of mouse bone marrow erythroid cells at various stages of differentiation was studied by using a change in buoyant density with pH as an index of swelling. The ability to swell with a pH drop was acquired while the cell was still nucleated. All non-nucleated cells showed swelling. Most small erythroblasts shared this property, whereas most large erythroblasts did not. 3. The density shift with pH was used to provide a purification scheme specific for erythroid cells. The bone marrow cells were first centrifuged to equilibrium in an isotonic albumin density gradient at neutral pH. Regions of the gradient containing the erythroid cells were collected, and the cells were recovered and redistributed in an albumin gradient at acid pH. The erythroid cells showed a specific density shift which removed them from contaminants. Preparations containing 90–97% erythroblasts were obtained by this technique. 4. Differentiation within the erythroid series was accompanied by a general increase in cell buoyant density at neutral pH. This density increase may have been a discontinuous process, since erythroid cells appeared to form a number of density peaks. 5. The pH shift technique, in association with established density distribution and sedimentation velocity procedures, provides a range of cell separation techniques for biological or biochemical studies of erythroid cell differentiation in the complex cell mixtures in bone marrow or spleen.