Cloning of complete genes for novel hydrolytic enzymes from Antarctic sea water bacteria by use of an improved genome walking technique

The increased demand for enzymes with new properties makes indispensable the development of easy and rapid strategies to obtain complete genes of new enzymes. Here a strategy is described which includes screening by PCR of new subtilases mediated by Consensus-Degenerate Hybrid Oligonucleotide Primers (CODEHOP) and an improved genome walking method to obtain the complete sequence of the identified genes. Existing methods of genome walking have many limitations, which make them inefficient and time consuming. We have developed an improved genome walking method with novel advances to get a simple, rapid and more efficient procedure based on cassette-ligation. Improvements consist basically in the possibility of a genomic DNA digestion with any restriction enzyme, blunting and 3' adenylation of digested DNA by Taq DNA polymerase to avoid self-circularization, followed by TA ligation of the adenine 3' overhanging end to the same unphosphorylated oligo-cassette. The efficiency of the genome walking method was demonstrated by finding the unknown ends of all gene fragments tested, previously obtained by CODEHOP-mediated PCR, including three subtilases (P4, P6 and P7), one xylanase and one lipase, from different strains of Antarctic marine bacteria.     

In vivo selection of protease cleavage sites by using chimeric Sindbis virus libraries

Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.     

The right-handed slanting of Arabidopsis thaliana roots is due to the combined effects of positive gravitropism,circumnutation and thigmotropism

When primary Arabidopsis roots grow down a tilted agar plate, they do not elongate following the gravitational vector along a straight line, but instead they slant noticeably to the right-hand. This process is seen mostly in the ecotypes Wassilewskjia and Landsberg, whereas it is attenuated in the ecotype Columbia, and in some mutants is even inverted. The origin of the slanting of Arabidopsis roots, that evidently constitutes a form of chirality, has so far not been sufficiently clarified. In the present paper we describe it as the general result of the cumulative effects of positive gravitropism, circumnutation and a thigmotropic obstacle-avoiding movement, and in particular, as the consequence of the alternating movement of circumnutation of the root to the right and to the left of the gravitational vector. This movement, which does not appear symmetrical in its nature, since the waves made to the right-hand are complete whereas those made to the left-hand are reduced or aborted, appears to be the reason for the observed slanting. In addition, evidence is furnished supporting the hypothesis that the strong left-handed cell-files torsion, seen in right-handed coiling roots, is not the consequence of a primary process, but of an artifact, and is due to the adjustment of the three dimensional root circumnutating helix to the flat two dimensional agar surface.     

On the Nature and Origin of the Calcium Asymmetry Arising during Gravitropic Response in Etiolated Pea Epicotyls

Seven day old etiolated pea epicotyls were loaded symmetrically with ³H-indole 3-acetic acid (IAA) or ⁴⁵Ca²⁺, then subjected to 1.5 hours of 1g gravistimulation. Epidermal peels taken from top and bottom surfaces after 90 minutes showed an increase in IAA on the lower side and of Ca²⁺ on the upper side. Inhibitors of IAA movement (TIBA, 9-hydroxyfluorene carboxylic acid) block the development of both IAA and Ca²⁺ asymmetries, but substances known to interfere with normal Ca²⁺ transport (nitrendipine, nisoldipine, Bay K 8644, A 23187) do not significantly alter either IAA or Ca²⁺ asymmetries. These substances, however, are active in modifying both Ca²⁺ uptake and efflux through oat and pea leaf protoplast membranes. We conclude that the ⁴⁵Ca²⁺ fed to pea epicotyls occurs largely in the cell wall, and that auxin movement is primary and Ca²⁺ movement secondary in gravitropism. We hypothesize that apoplastic Ca²⁺ changes during graviresponse because it is displaced by H⁺ secreted through auxin-induced proton release. This proposed mechanism is supported by localized pH experiments, in which filter paper soaked in various buffers was applied to one side of a carborundum-abraded epicotyls. Buffer at pH 3 increases calcium loss from the side to which it is applied, whereas pH 7 buffer decreases it. Moreover, 10 micromolar IAA and 1 micromolar fusicoccin, which promote H⁺ efflux, increase Ca²⁺ release from pea epicotyl segments, whereas cycloheximide, which inhibits H⁺ efflux, has the reverse effect. We suggest that Ca²⁺ does not redistribute actively during gravitropism: the asymmetry arises because of its release from the wall adjacent to the region of high IAA concentration, proton secretion, and growth. Thus, the asymmetric distribution of Ca²⁺ appears to be a consequence of growth stimulation, not a critical step in the early phase of the graviresponse.