Inter- and intra-population variations in diapause attribute of the two-spotted spider mite,Tetranychus urticae Koch,in Japan

Populations of the two-spotted spider mite, Tetranychus urticae Koch collected from various localities and from various host plants in Japan showed wide variations in diapause attribute. Diapause percentages at 18°C/9L15D varied from nearly 100% in the north to 0% in the south-west. At intermediate latitudes the mites showed wide inter-population variations. Populations on herbaceous hosts in vinyl- or glass-houses gave significantly lower incidence of diapause than those on roses and deciduous fruit trees. Presence of winter hosts and better host quality under protected environments seemed to favour non-diapausing mites. The temperature threshold for diapause expression also varied widely among local populations. Northern populations consistently had higher and less variable thresholds than populations at intermediate latitudes with thresholds between 15 and 18°C. Inbred lines derived from a population in Kyoto exhibited a wide variation in diapause percentage at 18°C. These results show that diapause in T. urticae is a quantitative threshold trait and that populations in central Japan consist of a variety of genotypes with different diapause traits. This might provide a genetic source for adaptation to local and temporal variations in environmental conditions.     

Autoregulation of the stability operon of IncFII plasmid NR1.

The stb locus of IncFII plasmid NR1, which mediates stable inheritance of the plasmid, is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins. The two tandem genes, stbA and stbB, are transcribed as an operon from promoter PAB. Using PAB-lacZ gene fusions, it was found that the stb operon is autoregulated. A low-copy-number stb+ plasmid introduced into the same cell with the PAB-lacZ fusion plasmid repressed beta-galactosidase activity about 5-fold, whereas a high-copy-number stb+ plasmid repressed beta-galactosidase about 15-fold. The details of autoregulation were analyzed by varying the concentrations of StbA and StbB to examine their effects on expression from the PAB-lacZ fusion plasmid. StbB protein by itself had autorepressor activity. Although StbA protein by itself had no detectable repressor activity, plasmids that encoded both stbA and stbB repressed more effectively than did those that encoded stbB alone. Plasmids with a mutation in stbA had reduced repressor activity. One mutation in stbB that inactivated the stability function also reduced, but did not eliminate, repressor activity. Repressor activity of the mutant StbB protein was effectively enhanced by stbA. These results indicate that StbB serves two functions, one for stable inheritance and one for autoregulation of the stb operon, both of which may be influenced by StbA protein.     

Purification and some properties of an extracellular l-amino acid oxidase from Cellulomonas cellulans AM8 isolated from soil

Summary The soil isolate Cellulomonas cellulans AM8 produces an extracellular l-amino acid oxidase (L-AAO) with broad substrate specificity. The strain produced up to 0.35 unit (U)/ml of the extracellular L-AAO in a simple medium containing glycerol and yeast extract. The enzyme was easily purified up to 30 U/mg protein using Phenyl-Sepharose fast flow. The purified enzyme migrated as single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 55 kDa. On native PAGE the molecular mass was approx. 300 000 kDa, which may be due to aggregation. With the exception of glycine, proline, and threonine, all the amino acids normally constituting proteins were oxidized. The V _max values from 0.7 to 35.2 U/mg for aspartic acid and lysine, respectively, and the K _m values from 0.007 to 7.1 mm for cysteine and valine, respectively, were obtained at 25° C and pH 7.0 in oxygen-saturated solutions. The L-AAO had a pH optimum of 6.5–7.5. It was stable for several months at — 30° C and for some days at 35° C. Ferricyanide served as an electron acceptor with a V _max of 50 U/mg and K _m for 0.3 mm with phenylalanine as the substrate. Correspondence to: R. D. Schmid     

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genome modifications in protoplast-derived tobacco plants: Contents of repetitive DNA sequences

Plasticity of the tobacco genome was studied by testing the DNAs of protoplast-derived regenerants with three different repetitive DNA sequences by the method of quantitative DNA/DNA hybridizations. A large population of 91 regenerants belonging to 35 different protoclones was analysed and a high degree of heterogeneity in the contents of the different DNA repeats was detected. The contents of middle repetitive sequences of two types were more stable or changed in the same direction, while the highly repetitive sequence varied independently and displayed a significant reduction in comparison with the two other sequences. Comparing the variation within the subpopulations of plants of the same clonal origin and the variation among the protoclones led to a conclusion that the pre-existing DNA variability in the starting plant material and/or thein vitro stress during the very early stages of protoclone regeneration played a decisive role in the formation of modified genomes in regenerants.     

PAS IV, an integral membrane protein of mammary epithelial cells, is related to platelet and endothelial cell CD36 (GP IV).

PAS IV is a 78-kDa (bovine) to 80-kDa (human) integral membrane glycoprotein of unknown function which is found in mammary epithelial cells. We now report the purification of human PAS IV and native bovine PAS IV from the milk fat globule membrane (MFGM), a preparation of apical plasmalemma from epithelial cells of lactating mammary tissue. N-Terminal sequence analyses of human and bovine PAS IV revealed homology to the N-terminal sequence of the 88-kDa human endothelial and platelet glycoprotein CD36. The similarity of MFGM PAS IV to platelet CD36 was further established by immunoblots of purified platelet CD36 and MFGM PAS IV with MFGM PAS IV specific antiserum. The removal of N-linked oligosaccharides from PAS IV and CD36 by treatment with endoglycosidase F reduced the apparent Mr of both proteins to approximately 57,000. These data suggest that PAS IV and CD36 are similar if not identical polypeptides that undergo cell type specific glycosylation.     

Determination of an antisecretory trimethyl prostaglandin E2 analog in human plasma by combined capillary column gas chromatography—negative chemical ionization mass spectrometry

A method is described for measuring a trimethyl prostaglandin E₂ analog, TM-PGE₂, in human plasma. Trideuterated and monofluorinated analogs of TM-PGE₂ are added to plasma as internal standard and carrier, respectively. The plasma is adjusted to pH 3.0 and is extracted with a mixture of benzene—dichloromethane (9:1). The residue, following removal of the extracting solvent, is reacted consecutively with pentafluorobenzyl bromide and bistrimethylsilyltrifluoroacetamide. The excess derivatizing reagents are removed by evaporation, and an aliquot of the reconstituted residue is analyzed by capillary column gas chromatography using methane as the carrier gas. A quadrupole mass spectrometer is set to monitor in the gas chromatographic effluent the (M − C₇H₂F₅)⁻ fragmention of TM-PGE₂ (m/e 449) and trideuterated TM-PGE₂ (m/e 452) generated by methane negative chemical ionization. Quantitation of unknowns is based on a comparison of the m/e 449 to m/e 452 ion ratio in each unknown to that obtained from the analysis of control plasma spiked with known amounts of TM-PGE₂ and fixed amounts of internal standard and carrier. The sensitivity limit of the assay is approximately 100 pg ml⁻¹, which is equivalent to 1 pg injected. The assay was used to measure the concentration of TM-PGE₂ in the plasma of two subjects following a single 10 μg kg⁻¹ oral dose of the drug.