Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA

A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32% versus 8%) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36% of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12% of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmetamorphic juveniles was usually less than 0.1 (range 0.05-50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment

A Classification of Minimal Sets of Torus Homeomorphisms

We provide a classification of minimal sets of homeomorphisms of the two-torus, in terms of the structure of their complement. We show that this structure is exactly one of the following types: (1) a disjoint union of topological disks, or (2) a disjoint union of essential annuli and topological disks, or (3) a disjoint union of one doubly essential component and bounded topological disks. Periodic bounded disks can only occur in type 3. This result provides a framework for more detailed investigations, and additional information on the torus homeomorphism allows to draw further conclusions. In the non-wandering case, the classification can be significantly strengthened and we obtain that a minimal set other than the whole torus is either a periodic orbit, or the orbit of a periodic circloid, or the extension of a Cantor set. Further special cases are given by torus homeomorphisms homotopic to an Anosov, in which types 1 and 2 cannot occur, and the same holds for homeomorphisms homotopic to the identity with a rotation set which has non-empty interior. If a non-wandering torus homeomorphism has a unique and totally irrational rotation vector, then any minimal set other than the whole torus has to be the extension of a Cantor set.Comment: Published in Mathematische Zeitschrift, June 2013, Volume 274, Issue 1-2, pp 405-42

Structure and tribological performance of diamond-like carbon based coatings for aerospace component processing

Copyright @ 2009 The Surface Science Society of JapanThis work examines diamond-like carbon (DLC) coatings deposited by plasma enhanced chemical vapour deposition (PECVD) as an environmentally friendly alternative to chromium plating in restoration of worn or damaged aircraft components. DLC coatings offer superior mechanical properties; however, high internal stresses and poor adhesion can prevent the deposition of thick films. This work examines a series of layered structures based on epoxy-resin interlayers with DLC applied as a surface film. Wear testing and examination with scanning electron microscopy and atomic force microscopy lead to the development of an optimum DLC/epoxy system with wear characteristics superior to those of chromium-plated steel. This new coating system has a great potential in restoring aircraft components in a more efficient and environmentally friendly manner.This work is funded via the Engineering and Physical Sciences Research Council (EPSRC)

Negative spatial regulation of the lineage specific CyIIIa actin gene in the sea urchin embryo

The CyIIIa·CAT fusion gene was injected into Strongylocentrotus purpuratus eggs, together with excess ligated competitor sequences representing subregions of the CyIIIa regulatory domain. In this construct, the chloramphenicol acetyltransferase (CAT) reporter gene is placed under the control of the 2300 nucleotide upstream regulatory domain of the lineage-specific CyIIIa cytoskeletal actin gene. CAT mRNA was detected by in situ hybridization in serial sections of pluteus stage embryos derived from the injected eggs. When carrier DNA lacking competitor CyIIIa fragments was coinjected with CyIIIa.CAT, CAT mRNA was observed exclusively in aboral ectoderm cells, i.e. the territory in which the CyIIIa gene itself is normally expressed (as also reported by us previously). The same result was obtained when five of seven different competitor subfragments bearing sites of DNA-protein interaction were coinjected. However, coinjection of excess quantities of either of two widely separated, nonhomologous fragments of the CyIIIa regulatory domain produced a dramatic ectopic expression of CAT mRNA in the recipient embryos. CAT mRNA was observed in gut, mesenchyme cells and oral ectoderm in these embryos. We conclude that these fragments contain regulatory sites that negatively control spatial expression of the CyIIIa gene

Competitive titration in living sea urchin embryos of regulatory factors required for expression of the CyIIIa actin gene

Previous studies have located some twenty distinct sites within the 2.3 kb 5' regulatory domain of the sea urchin CyIIIa cytoskeletal actin gene, where there occur in vitro high-specificity interactions with nuclear DNA-binding proteins of the embryo. This gene is activated in late cleavage, exclusively in cells of the aboral ectoderm cell lineages. In this study, we investigate the functional importance in vivo of these sites of DNA-protein interaction. Sea urchin eggs were coinjected with a fusion gene construct in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene is under the control of the entire CyIIIa regulatory domain, together with molar excesses of one of ten nonoverlapping competitor subfragments of this domain, each of which contains one or a few specific site(s) of interaction. The exogenous excess binding sites competitively titrate the available regulatory factors away from the respective sites associated with the CyIIIa.CAT reporter gene. This provides a method for detecting in vivo sites within the regulatory domain that are required for normal levels of expression, without disturbing the structure of the regulatory domain. We thus identify five nonoverlapping regions of the regulatory DNA that apparently function as binding sites for positively acting transcriptional regulatory factors. Competition with a subfragment bearing an octamer site results in embryonic lethality. We find that three other sites display no quantitative competitive interference with CyIIIa.CAT expression, though as shown in the accompanying paper, two of these sites are required for control of spatial expression. We conclude that the complex CyIIIa regulatory domain must assess the state of many distinct and individually necessary interactions in order to properly regulate CyIIIa transcriptional activity in development

Topological Entropy of Braids on the Torus

A fast method is presented for computing the topological entropy of braids on the torus. This work is motivated by the need to analyze large braids when studying two-dimensional flows via the braiding of a large number of particle trajectories. Our approach is a generalization of Moussafir's technique for braids on the sphere. Previous methods for computing topological entropies include the Bestvina--Handel train-track algorithm and matrix representations of the braid group. However, the Bestvina--Handel algorithm quickly becomes computationally intractable for large braid words, and matrix methods give only lower bounds, which are often poor for large braids. Our method is computationally fast and appears to give exponential convergence towards the exact entropy. As an illustration we apply our approach to the braiding of both periodic and aperiodic trajectories in the sine flow. The efficiency of the method allows us to explore how much extra information about flow entropy is encoded in the braid as the number of trajectories becomes large.Comment: 19 pages, 44 figures. SIAM journal styl

Comparison of Summer Forages and the Effect of Nitrogen Fertilizers on \u3ci\u3eBrassica\u3c/i\u3e Forages in Tasmania

Summer forage crops, and in particular Brassica spp., have become increasingly popular in dairy production systems in Tasmania. Field experiments were conducted for 3 years in northwestern Tasmania, in the spring/summers beginning in 1995. The study aimed to compare yield and quality of Brassica and Poaceae forages and the response of Brassica species to nitrogen (N) (50, 100 and 200 kg N/ha) and irrigation. The average total yields of dryland (rainfed) crops in 1995 to 1997 experiments, were turnip (Brassica rapa) 9.3 t/ha, rape (B. napus) 5.9 t/ha, oats (Aevena sativa) 5.2 t/ha, kale (B. oleracea) 5.1 t/ha, short-lived ryegrass (Lolium multiflorum) 5.1 t/ha, pasja (B. campestris × B. napus) 4.3 t/ha, perennial ryegrass (L. perenne) 4.2 t/ha, millet (Echinochloa utilis) 3.8 t/ha, and maize. (Zea mays) 2.9 t/ha. Irrigation increased the yield of turnips by 4.8 t/ha (mainly bulbs) and millet yields by 1.4 t/ha and reduced maize yield by 1.2 t/ha. Brassica species were higher in ME and lower in CP than the Poaceae forages. Nitrogen fertilizer increased the DM yield of tops of all Brassica crops in the 1997/98 experiments under irrigation, but it decreased the yield of turnips bulbs. The total yields with 50, 100 and 200 kg N/ha were 14, 15.2 and 15 t DM/ha for turnips, 7.5, 8.5 and 10 t for pasja and 10, 12 and 12.2 t DM/ha for rape, respectively. With 100 kg N/ha the average concentration of quality attributes of turnips, pasja and rape were CP 14, 22 and 19%, ME 12, 14.6 and 12.6 MJ/kg DM respectively. Nitrogen increased the CP, but had no effect on ME of any Brassica crops. Brassica forage are superior to Poaceae forages for summer feed production and as a part of pasture renovation process. They are higher in their yields, quality and water use efficiency and respond well to N fertilizer