Comparative study of the thiourea carrier in erythrocytes

Summary A densimeter technique was used to measure the rate of exit of thiourea from erythrocytes of various species of mammals. The cells were first equilibrated with a 200mm thiourea solution in 1% NaCl. An aliquot of these cells was added to 1% NaCl containing 4.6–23.1mm thiourea. Facilitated diffusion was demonstrated in each case. Using exit times or initial rates, calculations of half-saturation constants () inmm and maximum transport rates (K) in isotones per min were made by three different methods. The following values were obtained: human –=60, 42, 35;K=1.2, 2.9, 0.9; rabbit –=46, 33, 32;K=0.8, 2.1, 0.8; mouse –=46,40, 30;K=3.4, 8.5, 3.2; rat –=65, 42, 23;K=6.1, 15.3, 3.7; ox –=107, 63, 88;K=0.6, 1.4, 0.4; sheep –=56, 38, 56;K=0.9, 2.2, 0.6; and pig –=110, 64, 49;K=1.6, 3.6, 1.1.     

Intracellular effects of phage phi W-14 DNA on transformation of Bacillus subtilis

Summary Uptake of transforming DNA by competent Bacillus subtilis cells in the presence of phage W-14 DNA (in which half the thymine residues are replaced by -putrescinyl-thymine) is accompanied by a decrease in the amount of trichloracetic acid-precipitable label of the former retained by recipient cells during subsequent incubation. Fractionation of lysates of cells incubated for 0.5 min at 37°C after DNA uptake at 30°C in the presence of low concentrations of W-14 DNA (0.1 g/ml) demonstrated the presence of single-stranded transforming DNA molecules, typical for DNA taken up by B. subtilis. The intracellular effect of W-14 DNA was enhanced by an increase in its concentration (to 0.5–1 g/ml), or by increasing the temperature of uptake (to 37°C). With either of these treatments transforming DNA taken up was found in the form of a broad asymmetric band, indicative of degradation, and partially located at the density characteristic for single-stranded molecules. Fractionation of lysates of cells treated (0.1 g/ml) or untreated with W-14 DNA, and incubated for 20 min at 37°C after DNA uptake, showed disappearance of the single-stranded band. Donor DNA label was then found exclusively in the recipient DNA band, its amount being lower in samples treated with W-14 DNA. The influence of a high concentration of W-14 DNA on retention of transforming DNA label was correlated with its effect on transformation. On exposure to low concentrations of phage DNA, such a correlation was observed only after longer periods of incubation, due to slower intracellular degradation of homologous DNA taken up. The results are consistent with the proposal that W-14 DNA-induced reduction in efficiency of transformation is due to intracellular stimulation of transforming DNA degradation, leading to a decrease in the number of donor molecules available for recombination with the recipient chromosome.     

The distribution of Heterotrissocladius oliveri Saether (Diptera: Chironomidae) in Lake Michigan

Fifty one chironomid species were identified from 504 samples collected at depths ranging 8 to 267 m in Lake Michigan, U.S.A. Heterotrissocladius oliveri Saether occurred in 32% of these samples and had an average abundance of 22 m^–2 which was similar to other estimates from the Great Lakes. Maximum average lake-wide density was at 30 to 60 m (41 m^–2). At depths 60 m, H. oliveri was the dominant chironomid species comprising 75% of total Chironomidae. The substrate preference of H. oliveri differed within each depth regime considered: at 30–60 m, 2–3 ; at 60–120 m, 3–5 , 7–9 ; and at 120–180 m, 6–8 . Abundance was notably reduced at all depths in substrates characterized as medium silt (5–6 ). On a lake-wide basis, the distribution pattern suggested H. oliveri was most numerous from 30 to 60 m along the southwestern, eastern, and northern shorelines and at 60–120 m depths along the southern and eastern shorelines. Increased abundance in the South Basin was concurrent with evidence of increased sedimentation at 60 to 100 m. However, in several other areas of the lake, high densities were associated with medium to very fine sands relatively free of silts and clays. This observation suggested occurrence of H. oliveri was minimally affected by sediment type.Widely variable, but generally elevated water temperatures likely prevent H. oliveri from establishing a substantial population density at depths < 30 m. With increased depth, temperature fluctuation is negligible and food is more stable, though the source is variable. Factors limiting abundance of H. oliveri at depths 30 m were related to decreased food supply due to distance from shore, food sources of lower value (clays), and, most importantly, to reproductive replenishment.Although still oligotrophic in nature, high density occurrences in both high and low sedimentation areas of the lake suggest the trophic indicator status of H. oliveri might be broader than previously thought.     

Movement and compartmentation of abscisic acid in guard cells of Valerianella locusta: Effects of osmotic stress,external H+-concentration and fusicoccin

Epidermal peels of Valerianella locusta were acid-treated for 1 h at pH 3.9 to kill all cells other than guard cells. These guard-cell preparations were used to explore the steady-state one-way fluxes and the cytoplasmic and vacuolar contents of abscisic acid (ABA). The method of compartmental analysis has been applied. The intracellular ABA concentrations were surprisingly high. At an external pH of 5.8 the cytoplasm contained 1.28 mmol·dm^-3 of ABA, twice of the amount which accumulated in the vacuoles (0.57 mmol·dm^-3). The fluxes of ABA at the plasmalemma (_oc=_oc=0.43 fmol · cell ^–1 · h ^–1) were higher than those at the tonoplast (_cv=_vc=0.12 fmol · cell ^–1 · h ^–1). Moderate stress (0.1 and 0.3 mol·dm^-3 sorbitol in the medium) caused a change in the kinetics of ABA movement. The rate constants of the fluxes from the cytoplasm into the vacuole (_cv) and into the apoplast (_co) were increased while the rate constant of the flux from the vacuoles into the cytoplasm (_vc) was decreased. As a consequence the amount of ABA sequestered in the vacuole remained unchanged; the cytoplasmic ABA content, however, was reduced to only 20% of that found in the control treatments (no sorbitol in the medium). Under moderate stress, one Valerianella guard cell released rapidly about 0.36 fmol·cell^-1 to its direct cell-wall space. This surprising result is discussed in regard to rapid stomatal closure under reduced water supply.Abbreviations ABA abscisic acid - FC fusicoccin     

Restriction and modification in Bacillus subtilis 168. Regulation of hsrM(nonB) expression in spoOA mutants and effects on permissiveness for phi15 and phi105 phages

Summary Gene hsrM (nonB) of Bacillus subtilis 168, causing non-permissiveness to phage SP10 (Saito et al. 1979) and reduced plating efficiency of unmodified phage 105, is responsible for non-permissiveness of B. subtilis 168 for phages 15 and PZA. Upon transformation to sporulation deficiency (allele spoOA) B. subtilis 168 becomes permissive for 15 and PZA and loses the ability to restrict 105. spoOA str-1 double transformants of B. subtilis 168, however, retain the restriction 168 and non-permissiveness for 15 and PZA phages, in spite of their Spo^– phenotype. Therefore it appears that a functional product of the spoOA gene is required for expression of gene hsrM in wild-type bacteria, but is not essential in streptomycin-resistant bacteria. Phage genomes (PZA) were trapped in spores of the restriction deficient strain with much higher efficiency than in the wild-type.     

Existence and uniqueness of a sharp travelling wave in degenerate non-linear diffusion Fisher-KPP equations

In this paper we use a dynamical systems approach to prove the existence of a unique critical value c ^* of the speed c for which the degenerate density-dependent diffusion equation u _ct = [D(u)u _x ] _x + g(u) has: 1. no travelling wave solutions for 0 < c < c ^*, 2. a travelling wave solution u(x, t) = (x - c ^* t) of sharp type satisfying (– ) = 1, () = 0 ^*; '(^*–) = – c ^*/D'(0), '(^*+) = 0 and 3. a continuum of travelling wave solutions of monotone decreasing front type for each c > c ^*. These fronts satisfy the boundary conditions (– ) = 1, '(– ) = (+ ) = '(+ ) = 0. We illustrate our analytical results with some numerical solutions.     

Transductional mapping of nine linked chromosomal genes in Bacillus thuringiensis

Summary We have previously described a phage (63) for generalized transduction in Bacillus thuringiensis and used it for mapping of four chromosomal antibiotic resistance markers, namely nalA-rifA-strA-spcA (Landén et al. 1981). From 63 we have now isolated a host range mutant called 64 which contains 52–56 megadalton of DNA. Phage 64 was found to be a more efficient transducing vector than 63. The host range of 64 is wide, with good growth on subspecies gelechiae, kurstaki, galleriae, thuringiensis and thompsoni, restriction on some derivatives of finitimus and ostrinae and no growth on alesti, israelensis and aizawai.Using 64 and a series of new mutants of subspecies gelechiae we have no added five new genes to the antibiotic resistance group described before. The gene order found was guaB-purB-metA-novA-(purA-nalA)-rifA-strA-spcA. Linkage was also demonstrated between hisA and lysA.     

Characterization of the effectors required for stable inheritance of Streptococcus pyogenes pSM19035-derived plasmids in Bacillus subtilis

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (or) [ and ]. Inactivation or deletion of or results in SegA^– plasmids. Better than random segregation requires an active segB region. The segB region contains two ors (or and or). Inactivation of either of the orfs does not lead to an increase in cell death, but or^– plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.     

Yeast ribosomal proteins

Summary Homology maps between bacteriophages 81, 80 and were constructed on the basis of electron microscope observation of DNA heteroduplexes. In 81/80 heteroduplex, the left half and the right terminal region of 13% the total molecular length were highly homologous, while the remaining region covering the early gene cluster was entirely nonhomologous. In 81/ heteroduplex, high-degree homologies were detected at the left 14% terminal region covering the head gene cluster, the central 3.8% region covering the att-int-xis region and the 1.3% Q homology region. Low-degree homologies of shorter length were scattered at the tail gene cluster, b2 region, cIII region, PQ region and SR region. Comparing our results with the homology maps of other lambdoid phages reported by Simon et al. (1971) and Fiandt et al. (1971), a phylogenic relation of 81 to other lambdoid phages and the role of recombination in the course of divergence of lambdoid phages are discussed.     

Bacteriophage phi 1 as a gene-cloning vector in Bacillus subtilis

Summary We attempted to use Bacillus subtilis phage 1 as a gene-cloning vector since the 1 genome was found to have few cleavage sites upon digestion with several kinds of restriction endonucleases. A 1 stock supplied by J. Ito (University of Arizona, Tucson, USA) consisted of two phages, 1E1 and 1E2, having one and two EcoRI-cleavage sites in their genomes respectively. From the latter isolate a deletion mutant 1E21 was induced to increase the size range of DNA segments to be cloned. It was demonstrated, by in vitro recombination experiments with phage 11 DNA, that 1E21 can be used for cloning EcoRI fragments of various sizes. We analyzed the DNAs of ten 1 clones isolated from independent transfectants and found that six of them carried 11 DNA fragments inserted at either of the two EcoRI-cleavage sites. Some of the hybrid phage DNAs were found to be cleaved with BamHI and HaeIII endonucleases at the 11 DNA portion, whereas the parental 1E21 DNA was insensitive to any of these enzymes. These hybrid phages would therefore be useful vectors for cloning foreign DNA fragments generated by cleavage with BamHI or HaeIII endonucleases.     

Sensitivity of Escherichia coli to viral nucleic acid. VI. Capacity of dna mutants and DNA polymerase-less mutants for multiplication of phiA and phiX 174

Summary Seven groups of dna mutants were tested for the capacity to support the growth of A and X174, using a calcium-dependent transfection system. At the restrictive temperature, two groups of mutants, dnaA and dnaF, allowed the viral multiplication. Group B, C, D, E and G mutants were nonpermissive at 43°C to SS¹ DNA as well as to double-stranded RF molecule. Evidence showing the dispensability for the viral growth of DNA polymerase I and recombination function was also presented. Double mutant deficient in DNA polymerase I and II supported the growth of A sufficiently.     

Transfection with replicating DNA from the temperate Bacillus bacteriophage ?105 and with T4-ligase treated ?105 DNA: The importance in transfection of being longer than genome-length

Summary Replicating phage DNA extracted from Bacillus subtilis infected with phage 105 has a higher activity in transfection than mature DNA. By heteroduplex analysis it was shown that this DNA contains concatemeric molecules. Concatemers, constructed in vitro by treatment of mature DNA with T4-ligase also have an increased activity in transfection. DNA showing an increased activity in transfection does not have a requirement for more than one molecule per transfection event as is typically found for transfection with mature 105 DNA. An explanation is given for this difference suggesting that the structure of the ends of the transfecting molecules play an important role in transfection.     

Root biomass changes after drainage and fertilization of a low-shrub pine bog

The stump and root systems of Scots pine (Pinus sylvestris) and field-layer vegetation were sampled before (1984) and three growing seasons after drainage and fertilization (1987) of a low-shrub pine bog. Average below-ground biomass of the field layer was 548 gDW m^–2 in 1984, with no significant treatment effects during experimentation. The stump-plus-root biomass of the pine stands was 1464 gDW m^–2 in the virgin state, and had increased to 1854 gDW m^–2 three years after the NPK-fertilizer treatment. The distribution over fractions also changed with this treatment. The fraction of fine roots ( < 1 mm) in stump-root biomass increased from 4% (56 gDW m^–2) to 11% (196 gDW m^–2), while the other compartments changed less. Total pine root length was 729 mm^–2 in 1984. Root length increased by 94% to 1380 mm^–2 on NPK-fertilized plots. Most of the fine pine roots were in the surface layer (0–10 cm), 79% in 1984 and 88% in 1987, and few pine roots were deeper than 20 cm. Maximum root length of fine pine roots ( < 1 mm) was estimated to be 2710 mm^–2 at about 800 gDW m^–2 (NPK treatment), and the corresponding maximum for small pine roots (=1–10 mm) was 227 mm^–2 at 809 gDW m^–2. Drainage stimulated net growth of fine roots, but this treatment also caused higher mortality rates of small roots. The fine roots responded to fertilization with higher net growth rate, and secondary growth of the large roots ( > 10 mm) was improved. The observed changes in root biomass and structure are explained as strategic adaptations to altered hydrological and nutritional circumstances in the root zone after drainage and fertilization.     

Transfection of protoplasts of Bacillus subtilis with ?29 DNA

Summary The protoplast-polyethyleneglycol(PEG) transformation procedure of Chang and Cohen (2) can be used for 29 DNA transfection. 29 DNA without terminal proteins is not transfectious in the protoplast-PEG procedure.     

The temperate B. subtilis phage phi 105 genome contains at least two distinct regions encoding superinfection immunity

Summary Two different PstI fragments of temperate phage 105 DNA are shown to confer superinfection immunity upon Bacillus subtilis when inserted into the multicopy cloning vector pE194 cop-6. The 2.3 kb PstI fragment I is located almost entirely within EcoRI fragment F and encompasses a region previously known to encode a repressor. The other fragment, PstI-E (4.3 kb) maps inside the EcoRI-B fragment, and allows an explanation of the clear-plaque phenotype of the deletion mutant 105DII:6c. The two regions can be distinguished functionally, since only the PstI fragment I product interacts with a specific 105 promoter-operator site.     

Upper limit for DNA packaging by Bacillus subtilis bacteriophage phi 105: isolation of phage deletion mutants by induction of oversized prophages

Summary We have determined the upper size limit for DNA packaging in Bacillus subtilis bacteriophage 105 by examining the plaque-forming and transducing capabilities of lysates made from strains containing prophages of various sizes. The upper size limit for efficient packaging of the phage genome appears to be about 40.2 kb, which is about 1 kb larger than the wild-type genome. This places an upper limit of about 5 kb on the size of insertions that can be accommodated in 105 transfection cloning vectors, such as 105J27. Induction of prophages that exceed the upper limit, followed by selection for plaque formation or transduction, provides a powerful means of isolating phage deletion mutants. A comparison of the location of each deletion with the resultant phenotype has enabled us to identify non-essential regions of the phage genome, and regions that are required for tail biosynthesis and for host cell lysis.     

DNA Replication and Cell Cycle Progression Regulatedby Long Range Interaction between Protein Complexes bound to DNA

A nonstationary interaction that controlsDNA replication and the cell cycle isderived from many-body physics in achemically open T cell. The model predictsa long range force F() =– (/2) (1 – )(2 – )between thepre-replication complexes (pre-RCs) boundby the origins in DNA, = /N being the relativedisplacement of pre-RCs, the number of pre-RCs, Nthe number of replicons to be replicated,and the compressibilitymodulus in the lattice of pre-RCs whichbehaves dynamically like an elasticallybraced string. Initiation of DNAreplication is induced at the threshold = N by a switch ofsign of F'(), fromattraction (–) and assembly in the G ₁ phase (0<<N), to repulsion (+) and partialdisassembly in the S phase (N< < 2N), withrelease of licensing factors from pre-RCs,thus explaining prevention ofre-replication. Replication is terminatedby a switch of sign of force at = 2N, from repulsion inS phase back to attraction in G ₂, when all primed replicons havebeen duplicated once. F(0) = 0corresponds to a resting cell in theabsence of driving force at = 0. The model thus ensures that the DNAcontent in G ₂ cells is exactlytwice that of G ₁ cells. The switch of interaction at the R-point, at which N pre-RCs have been assembled, starts the release of Rb protein thus also explaining the shift in the Rb phosphorylation from mitogen-dependent cyclinD to mitogen-independent cyclin E.Shape,slope and scale of the response curvesderived agree well with experimental datafrom dividing T cells and polymerising MTs,the variable length of which is due to anonlinear dependence of the growthamplitude on the initial concentrations oftubulin dimers and guanosine-tri-phosphate(GTP). The model also explains the dynamic instabilityin growing MTs.     

Plasmid transformation in Bacillus subtilis. The significance of partial homology between plasmid and recipient cell DNAs

Summary A series of hybrid plasmids consisting of pC194 or pUB112 and B. subtilis DNA were constructed. In contrast to plasmid pC194, purified monomeric forms of such plasmids were active in transformation, provided the recipient cells were recombination proficient. Similarly the monomers of PC194 derived plasmids, containing bacteriophage 105 DNA were able to transform 105 lysogenic but not nonylsogenic cells. From the results it is concluded that the presence of DNA/DNA homology between chromosomal DNA of the recipient cell and part of the hybrid plasmids used is a sufficient condition to endow monomeric plasmids with transforming activity.This work is part of the Doctoral Thesis to be submitted by A. Iglesias to the Freic Universität Berlin     

Isolation and characterization of deletion mutants affected in early genes of bacteriophage ?80

Summary When Escherichia coli cells that had been irradiated with ultraviolet light were infected with bacteriophage 80, five major (pE, pB, pA, pC and pD) and two minor (pU and pV) proteins were found to be synthesized during early stages of infection. The genss coding for the five major proteins were mapped on the 80 chromosome using various deletion mutants which lacked the capacity to synthesize some or all the major proteins. The size and positions of all the deletions were determined by gel electrophoresis of EcoRI digests of phage DNA and by electron microscopy of heteroduplexes between DNAs of the deletion and wild-type phage. The five major proteins designated pE(25K), pB(40K), pA(45K), pC(34K) and pD(31K) were shown to be encoded in this order presumably by a single operon that was located at 60.2–67.4% on the 80 genome. These proteins were found to be involved in phage recombination. The absence of pE or pB resulted in a Red^– phenotype and the absence of three proteins (pE, pB and pA) resulted in a Fec^– phenotype. The exact positions of the genes for the minor proteins pU(29K) and pV(26K) have not been determined.     

On the dose response in B. subtilis transfection: involvement of aggregates in phi 29 transfection.

Summary Transfection with 29 DNA is predominantly caused by multimolecular, protease-sensitive aggregates of DNA. A minority of transfecting DNA molecules having properties of unit-length 29 genome molecules show a quadratic dose response in transfection.