High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.     

Processing of behaviorally relevant temporal parameters of acoustic stimuli by single neurons in the superior olivary nucleus of the leopard frog

Summary Response characteristics of 130 single neurons in the superior olivary nucleus of the northern leopard frog (Rana pipiens pipiens) were examined to determine their selectivity to various behaviorally relevant temporal parameters [rise-fall time, duration, and amplitude modulation (AM) rate of acoustic signals. Response functions were constructed with respect to each of these variables. Neurons with different temporal firing patterns such as tonic, phasic or phasic-burst firing patterns, participated in time domain analysis in specific manners. Phasic neurons manifested preferences for signals with short rise-fall times, thus possessing low-pass response functions with respect to this stimulus parameter; conversely, tonic and phasic-burst units were non-selective and possessed all-pass response functions. A distinction between temporal firing patterns was also observed for duration coding. Whereas phasic units showed no change in the mean spike count with a change in stimulus duration (i.e., all-pass duration response functions), tonic and phasic-burst units gave higher mean spike counts with an increase in stimulus duration (i.e., primary-like high-pass response functions). Phasic units manifested greater response selectivity for AM rate than did tonic or phasic-burst units, and many phasic units were tuned to a narrow range of modulation rates (i.e., band-pass). The results suggest that SON neurons play an important role in the processing of complex acoustic patterns; they perform extensive computations on AM rate as well as other temporal parameters of complex sounds. Moreover, the response selectivities for rise-fall time, duration, and AM rate could often be shown to contribute to the differential responses to complex synthetic and natural sounds.Abbreviations SON superior olivary nucleus - DMN dorsal medullary nucleus - TS torus semicircularis - FTC frequency threshold curve - BF best excitatory frequency - PAM pulsatile amplitude modulation - SAM sinusoidal amplitude modulation - SQAM square-wave amplitude modulation - MTF modulation transfer function - PSTH peri-stimulus time histogram     

Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.     

The equine protease inhibitory system (Pi): Abnormal expressions of PiF,PiL, and PiS

Three cases of abnormal expression of the equine protease inhibitory alleles, Pi F, L, and S₁, were observed following the examination of 30,000 plasma samples by one-dimensional acid (pH 4.6) polyacrylamide gel electrophoresis. Characterization of the abnormal proteins in terms of isoelectric point, molecular mass, inhibitory spectra, and sialic acid content was performed using one- and two-dimensional electrophoretic techniques. The Pi F and S₁ abnormalities were postulated to be the result of amino acid substitutions causing alterations in the processing of the carbohydrate side chains. No explanation could be offered for the Pi L abnormality other than a charge shift mutation. Abnormal types, F*, L*, and S*₁ behaved as alleles but the distribution of L* in offspring from one stallion (present in only 6 of 83 offspring) differed significantly from expectation.This work was supported by a grant from the Australian Stud Book, Alison Road, Randwick, N.S.W. 2031.     

Identification of the peptide bond cleaved during activation of human Clr

CNBr cleavage of unreduced proenzyme Clr yielded fragment CP2b, isolated by gel filtration and highpressure gel permeation chromatography. This fragment (˜ M_τ 55000) comprised at least 4 disulphidelinked peptides, which were separated by gel filtration after reduction and alkylation. Peptide CP2bRA4, overlapping the A- and B-chain regions in proenzyme Clr was digested by V8 staphylococcal protease, and the digest separated by reversed-phase HPLC. N-terminal sequence analysis of peptide CP2bRA4SP9 established that Clr activation involves the cleavage of a single Arg-Ile bond, located in the sequence: Gln-Arg-Gln-Arg-Ile-Ile-Gly-Gly     

Crystal structure of subtilisin complexed with its trapped substrateStreptomyces subtilisin inhibitor—Protein-protein interaction and evolution of serine proteinases and their proteinaceous inhibitors

The complex of a bacterial alkaline serine proteinase, subtilisin BPN’, with its proteinaceous inhibitorStreptomyces subtilisin inhibitor is unique in several respects, compared with other similar complexes containing serine proteinases of trypsin family. In addition to the usual antiparallelβ-sheet involving P₁-P₃ residues of the inhibitor, P₄-P₆ residues form antiparallelβ-sheet with a previously unnoticed chain segment (the ‘S4-6 site’) of subtilisin. The ‘S4-6 site’ does not exist in serine proteinases of trypsin family, whether of mammalian or microbial origin. Global induced-fit movement seems to occur on the ‘trapped substrate’Streptomyces subtilisin inhibitor: a channel-like structure in SSI remote from the contact region becomes about 2 Å wider upon complexing with subtilisin. Main role of the secondary contact region ofStreptomyces subtilisin inhibitor seems to support the reactive site loop (primary contact region). Steric homology for the two contact regions is so high between the inhibitors ofStreptomyces subtilisin inhibitor family and those of pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family that it seems to favour a divergent evolution and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forwarded by Doolittle(Nature (London),272, 581, 1978).     

Protease activities in non-embryogenic and embryogenic carrot cell strains during callus growth and embryo formation

Embryogenic and non-embryogenic cell strains of Daucus carota L. were examined for their protease activity using a wide range of chromogenic synthetic peptides as substrates. High arginine-specific activity was present in all strains, but no protease activity "specific" for embryogenic or non-embryogenic strains could be detected with the substrates tested. The specific protease activity was 5–10 times higher in the non-embryogenic as compared to the embryogenic strain for most tested substrates, and this difference was not due to release of proteases in the latter. All strains showed a decrease in protease activity when cultured in media without 2,4-dichlorophenoxyacetic acid, but the embryos had high protease activity in comparison with the nondifferentiated cell aggregates. In the latter aggregates, hydrolyzing activity towards three of the substrates (H-D-Phe-Pip-Arg- p -nitroanilide, Suc-Ala-Pro-Phe- p -nitro-anilide and Bz-Phe-Val-Arg- p -nitroanilide) was absent, whereas the embryos were able to hydrolyze them.     

Mode of high temperature injury to wheat during grain development

High temperature stress adversely affects wheat growth in many important production regions, but the mode of injury is unclear. Wheat ( Triticum aestivum L. cv. Newton) was grown under controlled conditions to determine the relative magnitude and sequences of responses of source and sink processes to high temperature stress during grain development. Regimes of 25°C day/15°C night, 30°C day/20°C night, and 35°C day/25°C night from 5 days after anthesis to maturity differentially affected source and sink processes. High temperatures accelerated the normal decline in viable leaf blade area and photosynthetic activities per unit leaf area. Electron transport, as measured by Hill reaction activity, declined earlier and faster than other photosynthetic processes at the optimum temperature of 25/15 °C and at elevated temperatures. Changes in RUBP carboxylase activities were similar in direction but smaller in magnitude than changes in photosynthesic rate. Increased protease activity during senscence was markedly accentuated by high temperature stress. Specific protease activity increased 4-fold at 25/15 °C and 28-fold at 35/25 °C from 0 to 21 days after initiation of temperature treatments. Grain-filling rate decreased from the lowest to the highest temperature, but the change was smaller than the decrease in grain-filling duration at the same temperatures. We concluded that a major effect of high temperature is acceleration of senescence, including cessation of vegetative and reproductive growth, deterioration of photosynthetic activities, and degradation of proteinaceous constituents.     

Neural mechanisms of auditory information processing by identified interneurones in Orthoptera

Three qualities of sound—the directional, the temporal and the spectral—are important for intraspecific communication in Orthoptera. The neural mechanisms employed by identified interneurones for encoding these sound qualities are illustrated by examples of physiological processes found at different levels of the CNS. Discussed are: (1) the creation of directional information by local interneurones in the thorax, and the use of time-intensity trading in sound location; (2) mechanisms for encoding the temporal parameters of sound by interneurones ascending to the brain; (3) frequency-dependent neural filtering of auditory information by local interneurones.