Claude Potvin

Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli

SummarySeveral hundred bacterial isolates were screened for bacteriolytic activity by growing them on agar medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells as the substrate. A Bacillus sp. producing the largest lytic zone was selected. A genomic bank of this selected bacterium was constructed in the multi-functional vector pTZ18R, with partial SauIIIA DNA fragments inserted at the SalI restriction site. Screening of 800 colonies of this bank for cell lysis gave 5 recombinants exhibiting lytic activity, as detected by analysis of extracts of sonicated Escherichia coli cells on denaturing polyacrylamide gels containing autoclaved, lyophilized M. lysodeikticus cells as the substrate. One clone (pBH2500), expressed inE. coli strain NM522, was found to code for a lytic enzyme corresponding, in molecular weight, to the 27 kDa Bacillus sp hydrolase. This clone with an insertion of 2.5 kb was then subcloned as a 929 bp EcoRI-SauIIIA fragment in pTZ18R (pBH929) and showed higher cell lytic activity. A unique open reading frame for a protein of 251 amino acids, followed by a putative terminator sequence, was found after a consensus ribosome binding site. A putative leader sequence was identified in the first 37 amino acids. One truncated subclone (pBH703), corresponding to 196 out of 251 residues from the protein N-terminal end, still possessed lytic activity.

Secreted hen lysozyme in transgenic tobacco: recovery of bound enzyme and in vitro growth inhibition of plant pathogens

Abstract Less than 1% of secreted hen egg white lysozyme (HEWL) was isolated in intercellular fluid neutral phosphate buffer extracts of transgenic tobacco ( Nicotiana tabacum L.). When foliar tissue was infiltrated with 50 mM calcium chloride or 1% histamine, 10–20% of HEWL was isolated when compared to HEWL obtained in tissue homogenates boiled in the presence of sodium dodecyl sulfate. The presence of mature HEWL was shown by N-terminal amino acid microsequencing of the major electrophoretic form (14.4 kDa). A minor electrophoretic form (17 kDa) having the same N-terminus end (KVFGRC) as the major mature from was also found. Tissue imprinting of transgenic tobacco cut stem, petiole and leaf tissue for detecting lysis of Micrococcus luteus cells embedded as lysozyme substrate in polyacrylamide gels could be performed in the presence of histamine for release of HEWL. Despite tight binding of HEWL in transgenic tobacco, some HEWL activity could be detected around growing transgenic tobacco seedling rootlets. Moreover, HEWL recovered from transgenic tobacco extracellular extracts was shown to inhibit the growth of some bacterial and fungal plant pathogens.

Expression of active hen egg white lysozyme in transgenic tobacco

A full-length complementary DNA clone of hen egg white lysozyme (HEWL) was inserted into pBI121 vector lacking the β-glucuronidase (GUS) gene and used to transform tobacco. The HEWL gene had its own 18-amino acid signal peptide. HEWL activity was detected using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay. The enzyme was active against Micrococcus luteus cells and migrated at the same molecular mass as purified mature HEWL. The protein was also found by immunodetection of HEWL after SDS-PAGE. Twenty-seven independent transgenic tobacco plants were analyzed for the expression of HEWL by a modified lysoplate assay for quantifying HEWL activity. Twenty-five plants exhibited HEWL activity up to 30 ng of HEWL per mg of leaf tissue. When using successive intercellular fluid (IF) extracts to study the localization of HEWL, it was found that no more than 10% of HEWL could be recovered in IF extracts. This is contrary to several extracellular proteins, such as the pathogenesis-related proteins. However, purified HEWL was recovered with the same yield as in transgenic tobacco leaves when it was injected into normal leaf tissue. Transgenic tobacco plants did not exhibit a phenotype change due to the expression of HEWL. The HEWL gene could be a useful reporter gene because the activity of the protein can be quantified by a simple assay.

Annual cycle of patterns of activity rhythms in beaver colonies (Castor canadensis)

SummaryBeavers studied under natural conditions near Québec City, Canada, displayed a yearly cycle of patterns of activity rhythms. In winter, the beaver colonies had a free-running circadian rhythm of period length 26.25 to 28.0 h, with or without relative coordination, depending on available light intensity, which in turn depends on ice and snow cover conditions; in summer, their activity rhythm followed a “normal” 24 h period. Transitions between these two patterns suggest that annual variations in the beavers physiological state affect their reaction towards the presence or absence of a Zeitgeber.

Androgen regulation of canine prostatic arginine esterase mRNA using cloned cDNA

Canine prostatic arginine esterase complementary DNA has been cloned in pPBS27, a new cloning vector. The relative abundance of androgen-regulated mRNA in intact dog prostate was reflected by the finding that a high proportion of the clones in the cDNA library hybridized strongly by plaque or colony hybridization with a poly(A)+ RNA probe from intact dog prostate but not with a poly(A)+ RNA probe from castrated dog prostate. One clone carrying a 400 base pairs cDNA insert was selected for further studies. Translation of the hybrid-selected RNA in a cell-free system resulted in the production of a 31 kDa peptide immunoprecipitable by antibodies against arginine esterase. This identification was confirmed by partial sequence analysis of the cDNA revealing an encoding protein with high homology to known kallikreins. Northern blot analysis of poly(A)+ and total RNA showed that arginine esterase mRNA had an approximate size of 1.0 kb which corresponded to a major androgen-regulated RNA species that could be observed after denaturing agarose gel electrophoresis of prostatic poly(A)+ RNA from intact dogs. Dot-blot analysis showed that dogs which had been castrated 3 weeks before had more than 100-fold lower arginine esterase mRNA level than intact dogs or castrated dogs treated with Depo-testosterone.

A Streptomyces chitosanase is active in transgenic tobacco

SummaryGrowth inhibition towards Rhizopus nigricans, Fusarium oxysporum f. sp. radicis-lycopersici, Verticillium albo-atrum and Pythium ultimum was observed in vitro using a purified chitosanase from an actinomycete, Streptomyces sp, strain N174. The corresponding gene, with its own signal peptide, was inserted into pBI121.7 shuttle vector to transform tobacco. Transgenic plants were analysed for chitosanase activity by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay. Two major and one minor active electrophoretic forms were detected in transgenic tobacco. Some chitosanases were recovered not only in leaf homogenates but also in leaf intercellular fluid extracts. One chitosanase electrophoretic form migrated very closely to the purified Streptomyces mature protein while the others corresponded to molecules of higher molecular mass. The N-terminus sequence was determined for one of the three chitosanase forms. It exhibited a different signal peptide cleavage site when compared to the mature chitosanase from Streptomyces. This is the first report on the expression of an active chitosanase gene with antimicrobial potential in plants.

HIGH-YIELD METHOD FOR DIRECTIONAL CDNA LIBRARY CONSTRUCTION

Improvement of a cDNA synthesis procedure using a single stranded (ss) vector primer [Bellemare et al., Gene 52 (1987) 11-19] is reported. This vector (pPBS27), upon linearization with XbaI using an appropriate restriction site-directed fragment, releases a thymidilic tail used to prime cDNA synthesis. DNA polymerase I and RNase H replace the RNA strand and replicate the vector before double-stranded (ds) blunt-end ligation with T4 DNA ligase. More than 10(7) cfu/microgram of vector can be obtained with an efficient transformation protocol using either globin-encoding or 7.5-kb poly(A)-tailed RNA. This improved cloning method is easier, faster and a few hundred times more efficient than the original procedure as it involves ds rather than ss DNA for transformation.

From Telecourses to Online Courses: A Story of Redesign | Du cours télévisé au cours en ligne : une histoire de redesign

This case deals with the redesign of a standard telecourse - printed material, professional studio video recordings and phone tutoring – into an online course. The redesign involved an adjunct professor in the Humanities having some experience in distance education but little with learning technologies. It was a two-year project including the grant application process. The main issues included replacing television-based content with multimedia content; understanding the complexity of interactions between materials, students, and tutors; and adapting traditional assessment approaches to online instruments and methods.