Chromosomal localization of a tandemly repeated DNA sequence in Trifolium repens L

INTRoDUCTIoNlYho1iumrePensL,whiteclover,isaneconomicallyimportantplantspeciesintemperatepastures.Asbrieflyreportedby[1],ithas16pairsofchromosomes(2n=32).Asyet,nodetailedcytologicalexaminationofthisspecies,suchasC-banding,hasbeenrep0rted.Inthelastdecade,thetechnique0fC-bandinghasbeenusedt0examinehighlyrepeatedsequencesinplantchrom0s0mesandhasprovidedausefultoolf0rtheanalysis0fcyt0geneticstructureincr0pplants[2-71.Inplants,thechr0m0s0mall0calizationofhighlyrepeatedDNAsequencesbyinsituhybr…     

Interactions between surfactants and biomass during liquid–liquid extraction

Fermentation systems can contain may surface‐active compounds that can interfere with downstream separation processes. This work examines the interactions that can occur between surfactants and biomass during solute mass transfer in a liquid–liquid extraction system. Adding the surfactants sodium dodecyl sulfate and dodecyl trimethyl ammonium bromide to the aqueous phase caused a substantial increase in the mass transfer of chloramphenicol between water and octanol. Further investigation of the interfacial region using an optical Schlieren apparatus revealed that these increases were due to interfacial turbulence that gave rise to a rapid surface renewal convective mass transfer mechanism. When microbial biomass was present with sodium dodecyl sulfate, an increase in the mass transfer rate was again found, however, to a lesser extent. In contrast, dodecyl trimethyl ammonium bromide did not promote mass transfer and it is postulated that electrical interactions between the surfactant and the cell surface prevented adsorption of either at the interface. The interaction between the antifoaming agent polypropylene glycol 2000 and extraction system components was also investigated, with both positive and negative effects being recorded under varying conditions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Mismatch repair-independent increase in spontaneous mutagenesis in yeast lacking non-essential subunits of DNA polymerase ε

Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3' to 5'exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system.     

Identification and cloning of two histone fold motif-containing subunits of HeLa DNA polymerase epsilon

HeLa DNA polymerase epsilon (pol epsilon), possibly involved in both DNA replication and DNA repair, was previously isolated as a complex of a 261-kDa catalytic subunit and a tightly bound 59-kDa accessory protein. Saccharomyces cerevisiae pol epsilon, however, consists of four subunits: a 256-kDa catalytic subunit with 39% identity to HeLa pol epsilon p261, a 80-kDa subunit (DPB2) with 26% identity to HeLa pol epsilon p59, a 23-kDa subunit (DPB3), and a 22-kDa subunit (DPB4). We report here the identification and the cloning of two additional subunits of HeLa pol epsilon, p17, and p12. Both proteins contain histone fold motifs which are present also in S. cerevisiae DPB4 and DPB3. The histone fold motifs of p17 and DPB4 are related to that of subunit A of the CCAAT binding factor, whereas the histone fold motifs found in p12 and DPB3 are homologous to that in subunit C of CCAAT binding factor. p17 together with p12, but not p17 or p12 alone, interact with both p261 and p59 subunits of HeLa pol epsilon. The genes for p17 and p12 can be assigned to chromosome locations 9q33 and 2p12, respectively.     

Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase epsilon

To better understand the functions and fidelity of DNA polymerase ε (Pol ε), we report here on the fidelity of yeast Pol ε mutants with leucine, tryptophan or phenylalanine replacing Met644. The Met644 side chain interacts with an invariant tyrosine that contacts the sugar of the incoming dNTP. M644W and M644L Pol ε synthesize DNA with high fidelity, but M644F Pol ε has reduced fidelity resulting from strongly increased misinsertion rates. When Msh6-dependent repair of replication errors is defective, the mutation rate of a pol2-M644F strain is 16-fold higher than that of a strain with wild-type Pol ε. In conjunction with earlier studies of low-fidelity mutants with replacements for the homologous amino acid in yeast Pol α (L868M/F) and Pol δ (L612M), these data indicate that the active site location occupied by Met644 in Pol ε is a key determinant of replication fidelity by all three B family replicative polymerases. Interestingly, error specificity of M644F Pol ε is distinct from that of L868M/F Pol α or L612M Pol δ, implying that each polymerase has different active site geometry, and suggesting that these polymerase alleles may generate distinctive mutational signatures for probing functions in vivo.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Double-blind Trial to Compare Ampicillin,Cephalexin, Co-trimoxazole,and Trimethoprim in Treatment of Urinary Infection

In order to test their value in urinary infection a double-blind trial was carried out using ampicillin, cephalexin, trimethoprim-sulphamethoxazole (co-trimoxazole), and trimethoprim. Eighty-three courses of treatment were given to hospital patients, 149 to pregnant women, and 107 to patients with dysuria and frequency seen in domiciliary practice. Thus infections of varying severity in defined groups of patients caused by organisms with different antibiotic sensitivities were treated.Analysis of the overall results (339 courses) was compared with those from the individual groups and considerable variation in response was found. In domiciliary infections and bacteriuria in pregnancy trimethoprim alone proved to be at least as effective as the other three compounds and caused fewer than half the number of side effects. In the hospital patients co-trimoxazole was superior to trimethoprim.The overall results for ampicillin and cephalexin were similar although cephalexin proved to be inferior in treating symptomatic domiciliary infections.     

Fissidens austro-americanus (Bryopsida: Fissidentaceae), a new species from Brazil and Suriname

Fissidens austro-americanus, from Brazil and Suriname, is described, illustrated, and its relationship toF. brevipes, F. ramicola, andF. leptophyllus discussed.     

Effect of fermentation broth and biosurfactants on mass transfer during liquid-liquid extraction

Mass transfer rates in liquid-liquid extraction processes can be seriously affected by the presence of surface-active contaminants. This is especially true of applications of a biotechnological origin, where the microorganism used in the process may produce the surface-active contaminants. An investigation into the effects of soluble and insoluble fermentation broth components on mass transfer using chloramphenicol extraction into octanol as the model system was conducted. Soluble components produced during fermentation were found to adsorb to the interface, where they reduced the overall mass transfer coefficient by up to 70%. After fractionation it was found that components in the weight range from 10-30 kDa had the greatest effect on mass transfer. Protein and phospholipid compounds of similar size were found to reduce the overall mass transfer coefficient to a similar extent to the broth components at concentrations around 0.001mg/l. The biomass produced during the fermentation also reduced mass transfer substantially, and it is likely that this was due to physical blockage of the interface.