Isolation and promoter characterization of barley geneItr1 encoding trypsin inhibitor BTI-CMe: differential activity in wild-type and mutantlys3a endosperm

The geneItr1, encoding trypsin inhibitor BTI-CMe, has been obtained from a genomic library ofHordeum vulgare L. The gene has no introns and presents in its 5-upstream region 605 bp that are homologous to the long terminal repeats (LTR) of the copia-like retro-transposon Bare-1. Functional analysis of theItr1 promoter by transient expression in protoplasts derived from different barley tissues, has shown that in this system theItr1 promoter retains its endosperm specifity and thetrans-regulation mediated by theLys3a gene. The proximal promoter extending 343 bp upstream of the translation initiation ATG codon is sufficient to confer fullGUS expression and for endosperm specifity. In protoplasts derived from thelys3a mutant, Risø 1508,GUS activity was less than 5% of that obtained with the same constructs in the protoplasts of wild-type Bomi from which it derives. Gel retardation experiments, after incubation with proteins obtained from both types of endosperm nuclei, also show differential patterns. Possible reasons for these differences are discussed.Equal authours     

Bacterial expression of an active class Ib chitinase from Castanea sativa cotyledons

Ch3, an endochitinase of 32 kDa present in Castanea sativa cotyledons, showed in vitro antifungal properties when assayed against Trichoderma viride. The characterization of a cDNA clone corresponding to this protein indicated that Ch3 is a class Ib endochitinase that is synthesized as a preprotein with a signal sequence preceding the mature polypeptide. Bacterial expression of mature Ch3 fused to the leader peptide of the periplasmic protein ompT resulted in active Ch3 enzyme. A plate assay was adapted for semi-quantitative determination of chitinase activity secreted from cultured bacteria, which should facilitate the identification of mutants with altered capacity to hydrolyse chitin.     

Solvent effects on the catalytic activity of subtilisin suspended in organic solvents

We studied a model transesterification reaction catalyzed by subtilisin Carlsberg suspended in toluene, n-hexane, diisopropyl ether, and mixtures of these solvents. To account for solvent effects due to differences in water partitioning between the enzyme and the bulk solvents, we measured water sorption isotherms for the enzyme in each solvent. We measured catalytic activity as a function of enzyme hydration and obtained bell-shaped curves with maxima at the same enzyme hydration in all the solvents. However, the activity maxima were different in all the media, being the lowest in toluene. Differences in the partitioning of substrates and product between the bulk solvent phase and the enzyme active site were accounted for but could not explain the lower catalytic activity observed in toluene. The fact that toluene is very similar to one of the substrates suggested the possibility of competitive inhibition by this solvent. We derived a model allowing for differences in solvation of the substrates, by using thermodynamic activities instead of concentrations, as well as for competitive inhibition by toluene. The model fit the experimental data well, confirming that toluene had a direct adverse effect on the catalytic activity of the enzyme. (c) 1996 John Wiley & Sons, Inc.     

Assessment of populations ofGracilaria chilensis (Graeilariales,Rhodophyta) utilizing RAPDs

Phenotypic variability and mixing of material due to massive cultivation for commercial purposes has contributed to the taxonomic confusion ofGracilaria in Chile. At least four species with cylindrical thalli and similar morphology have been recorded. However, since establishment ofG. chilensis, most of the collected thalli have been attributed to this species despite the lack of diagnostic features. In an attempt to resolve whetherGracilaria from 3 localities where it grows in natural and artificial populations belongs to the same species, gametophytic samples were compared by applying RAPD-PCR to their total DNA. This was analysed using 25 different 10-mer primers from which 21 revealed polymorphism within and between populations. Similarity matrices and cluster analyses were performed based on the presence/absence of bands representing fragments of DNA generated by random amplification. Similarity values between two of the populations were equivalent to those detected within a third, indicating the mixing of genetic material due to transplant between the two former localities. Similarities between samples of ChileanGracilaria andG. tenuistipitata from Sweden are considerably lower (0.45–0.53) than those between populations from Chile (0.74–0.88), confirming the existence of a single specific taxon,G. chilensis, in these three localities.     

Examining the relationship between secondary structure and antibody recognition in immunopeptides from foot-and-mouth disease virus

Summary The conformation of a peptide that represents antigenic site A of foot-and-mouth disease virus strain C-S8c1 (residues 136–156 of VP1; YTASARGDLAHLTTTHARHLP) has been studied by circular dichroism and compared with three analogs that reproduce amino acid substitutions at position 146 (HisArg, Gln or Asp) which affect antibody recognition. Four other peptides, incorporating replacements at position 147 predicted to maintain (LeuIle, Nle and Ala) or disrupt (LeuGly) helical structure at this site, have also been studied. In aqueous solution or in 4 M urea, the spectra of all eight peptides were typical of aperiodic conformation and independent of concentration or pH. However, upon addition of solvents such as methanol or hexafluoroisopropanol, spectral patterns evidenced significant levels (ca. 50%) of helical structure. The single residue substitutions at positions 146 and 147 caused minor to significant variations in the calculated amount of -helix of the peptides. An attempt to relate these changes in helical content to the antigenic behaviour of the peptides towards five monoclonal antibodies elicited with virus and mapping at site A could not find any straightforward correspondence between the two sets of results. The parent peptide and its His¹⁴⁶Arg analog were also analyzed by circular dichroism in the presence of the Fab fragment of SD6, a monoclonal antibody mapping at site A and much less reactive with viruses carrying the referred mutation. Although a peptide-antibody interaction was evident from spectral changes, careful inspection of the difference spectra (peptide-Fab minus Fab) of both peptides failed to detect any significant distinction between them that could be attributed to their different immunoreactivity. While these findings do not necessarily conflict with previous reports that the interaction of antigenic site A with antibodies is mediated to some extent by the adoption of a helix structure, they suggest that, at least for C-serotype viruses, other structural features in addition to a helical conformation are critically involved in antigenic recognition.     

Chaetomella acutiseta produces chaetomellic acids A and B which are reversible inhibitors of farnesyl-protien transferase

Chaetomellic acids A and B, isolated from Chaetomella acutiseta, are specific inhibitors of farnesyl-protein transferase that do not inhibit geranylgeranyl transferase type 1 or squalene synthase. Chaetomellic acids A and B are reversible inhibitors, resemble farnesyl diphosphate and probably inhibit FPTase by substituting for farnesyl diphosphate. Chaetomellic acid production appears to be widespread within the genus Chaetomella. Correspondence to: R. B. Lingham     

Biology of the bamboo Chusquea culeou (Poaceae: Bambusoideae) in southern Argentina

Over a period of 7 years the biology and phenotypic variability of Chusquea culeou were studied at 5 locations in cool temperate forests of southern Argentina. Excavated rhizomes had an average of 1.1 successful rhizome buds, and an average of 2.1 years elapsed between successive generations of rhizomes. Rhizome buds usually develop within the first four years after a rhizome forms. Height, volume and weight of a culm can be calculated from its diameter 1 m above the ground. Culm size, length of foliage leaf blades, and pattern of secondary branching differed among study sites. Dead culms were numerous and commonly remained erect for more than 7 years after dying. New culm shoots appear in spring and reach full size within a few months. Shoots can grow more than 9 cm/day. Less than half of the shoots survived a year; most were killed by moth larvae. Multiple primary branch buds emerge through the culm leaf sheaths in the second spring. The mean number of branch buds at mid-culm nodes varied between 34.8 and 81.5, and the mean number of primary branches was between 22.8 and 40.8. Number and length of branches, and number and length of foliage leaf blades at each node is related to the position of the node on a culm. Most branches grow about 3 cm and produce 1 to 3 foliage leaves annually. Foliage leaf blades generally live 2 years or more; few survive 6 years. Relative lengths of foliage leaf blades and their spacing along a branch permit recognition of annual cohorts.Both gregarious and sporadic flowering have been reported, and every year a few isolated plants flower and die. Length of the life cycle is unknown. Seedlings require up to 15 years to produce culms of mature size. Foliage branches may live more than 23 years, and culms may survive 33 years. Extensive loss of new shoots to predation suggests that gregarious flowering may be driven by a need to escape parasitism. C. culeou clumps expand slowly. Average annual rate of increase of the number of live culms in a clump was 4.6%. Methods of seed dispersal are undocumented. A dense stand of Chusquea culeou had an estimated phytomass of 179 tons/hectare (dry weight), 28% of which was underground. Net annual production was about 16 t/ha dry weight.