A simple,reliable rapid-mixing apparatus for continuous-flow studies

We have developed a simple and economical system for rapid mixing of solutions where a continuous-flow, chemically quenched mode of operation is appropriate. This has proved especially useful for studies of pyruvate dehydrogenase which produces a stable, enzyme-bound intermediate when the terminal acceptors are absent. It has also proved convenient for studies of the overall reaction in which fluorescence of pyridine nucleotide can be measured after quenching in alkaline solution.Syringes containing reactants are driven by a Harvard infusion pump, which provides sufficient speed and power (torque) to give good mixing with conventional commercially available mixing chambers and delay lines. Liquid chromatography fittings and valves are used for the switching operations necessary in the continuous-flow mode.     

Quantitation of DNA double-strand break resection intermediates in human cells

5′ strand resection at DNA double strand breaks (DSBs) is critical for homologous recombination (HR) and genomic stability. Here we develop a novel method to quantitatively measure single-stranded DNA intermediates in human cells and find that the 5′ strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle–dependent manner. Depletion of CtIP, Mre11, Exo1 or SOSS1 blocks resection, while depletion of 53BP1, Ku or DNA-dependent protein kinase catalytic subunit leads to increased resection as measured by this method. While 53BP1 negatively regulates DNA end processing, depletion of Brca1 does not, suggesting that the role of Brca1 in HR is primarily to promote Rad51 filament formation, not to regulate end resection.     

Tertiary educational assessment with mean individual level knowledge.

It is proposed that Tertiary Educational Assessment should be made using a Curve-Unifying Paradigm with its Scientific And Ultra-Conservative Experiment Ratio. Central Ranking Evaluation And Marking was used to process examination results, generating the Mean Individual Level Knowledge for the group. The concept of MILK grew from the need to encourage the average examination candidate and with it came the need for a Judgmental Understanding Goal. The results of some candidates required further handling by the addition of Student''s Universal Grade Averaging Regimen.     

Stimulation of V(D)J cleavage by high mobility group proteins.

V(D)J recombination requires a pair of signal sequences with spacer lengths of 12 and 23 bp between the conserved heptamer and nonamer elements. The RAG1 and RAG2 proteins initiate the reaction by making double-strand DNA breaks at both signals, and must thus be able to operate on these two different spatial arrangements. We show that the DNA-bending proteins HMG1 and HMG2 stimulate cleavage and RAG protein binding at the 23 bp spacer signal. These findings suggest that DNA bending is important for bridging the longer spacer, and explain how a similar array of RAG proteins could accommodate a signal with either a 12 or a 23 bp spacer. An additional effect of HMG proteins is to stimulate coupled cleavage greatly when both signal sequences are present, suggesting that these proteins also aid the formation of a synaptic complex.     

Production of ROL gene transformed plants of Rosa hybrida L. and characterization of their rooting ability

Transgenic plants of the rootstock Rosa hybrida L. cv. Moneyway were produced via a two-step procedure. First, kanamycin-resistant roots were generated on stem slices from micropropagated shoots, which were cocultivated with Agrobacterium tumefaciens containing the neomycin phosphotransferase II (NPTII) gene for conferring kanamycin resistance, together with individual ROL genes from A. rhizogenes. Root formation was quite efficient and up to two kanamycin-resistant roots per stem slice were produced. In the second step, these roots were used to regenerate transgenic plants via somatic embryogenesis. Although regeneration lasted up to 12 months, production of several transformants was successfully accomplished. Untransformed escapes were not found, indicating that the initial selection on kanamycin resistance was reliable.The presence of a combination of ROLA, B and C genes enhanced adventitious root formation on micropropagated shoots and explants of stems and leaves. It appears that the auxin sensitivity was increased to such a degree that cells were able to respond even to endogenous auxins present in shoots and leaves. Rooting experiments in greenhouse demonstrated that adventitious root formation on cuttings was improved threefold upon introduction of these ROL genes. It is concluded that a method was developed for the production of ROL gene transformed roses with improved rooting characteristics.     

Patterns of DNA and chromosome variation during in vitro growth in various genotypes of potato

Patterns of variation in nuclear DNA content and chromosome number were analysed in a temporal sequence, during in vitro growth of calli and cell suspensions in two monohaploids, a dihaploid and a tetraploid of potato (Solanum tuberosum). The results showed that both polyploidization and aneuploidy occurred during the initial stages of callus induction in all the genotypes. With further growth of callus, the frequency and extent of polyploidy and aneuploidy increased. In addition, the patterns of DNA and chromosome variation in cell suspension cultures revealed continued mitotic activity and transmission of cells with higher ploidy levels and aneuploidy. The results suggest that endoreduplication as well as endomitosis are important mechanisms of polyploidization, and that chromosome lagging and non-disjunction contribute to the production of aneuploidy.The various genotypes cultured under the same in vitro growth conditions differed in genetic instability, as assessed from the rate and degree of polyploidization and aneuploidy. Monohaploids showed more rapid rate of polyploidization than the dihaploid and tetraploid potatoes. It was concluded that the differences in genetic stability were due to different ploidy levels and genetic make-up of the genotypes.     

The hymenopteran parasitoids of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae) in Australia

Abstract  Epiphyas postvittana (Walker) is a serious pest of a number of horticultural crops including grapes in Australia and New Zealand. This study brings together information on the parasitoid complex associated with E. postvittana that previously was fragmented and largely inaccessible. We include species reared during a 3-year study of the parasitoids of E. postvittana in the vineyards of the Coonawarra region, South Australia, material from several Australian agricultural insect collections and records from the literature. An illustrated key is presented for 25 species of parasitoids and hyperparasitoids associated with E. postvittana , along with information on the taxonomy, identification, distribution and biology of each species. Taxa newly recorded from this host are Perilampus sp. (Perilampidae), and six species of Ichneumonidae: Euceros sp., Labium sp., Netelia sp., Plectochorus sp., Temalucha minuta (Morley) and Eriborus epiphyas sp. n., the latter species being described in full.