Group I introns are catalytic RNAs capable of self-splicing out of RNA transcripts. Ribozymes derived from these group I introns are used to explore the molecular recognition properties involved in intron catalysis. New ribozyme reactions are designed based on the inherent ability of these ribozymes to perform site-specific nucleophilic attacks. This study explores the molecular recognition properties of group I intron-derived ribozyme reactions and describe a new ribozyme reaction involving molecular recognition properties previously not seen.We report the development, analysis, and use of a new combinatorial approach to analyze the substrate sequence dependence of suicide inhibition, cyclization, and reverse cyclization reactions catalyzed by a group I intron from the opportunistic pathogen Pneumocystis carinii. We demonstrate that the sequence specificity of these Internal Guide Sequence (IGS) mediated reactions is not high, suggesting that RNA targeting strategies which exploit tertiary interactions could have low specificity due to the tolerance of mismatched base pairs.A group I intron-derived ribozyme from P. carinii has been previously shown to bind an exogenous RNA substrate, splice-out an internal segment, and then ligate the two ends back together (the trans excision-splicing reaction). We now report that a group I intron derived ribozyme from the ciliate Tetrahymena thermophila can also perform the trans excision-splicing reaction, although not nearly as well as the P. carinii ribozyme.In addition, we discovered a new ribozyme reaction called trans insertion-splicing where the P. carinii ribozyme binds two exogenous RNA substrates and inserts one directly into the other. Although this reaction gives the reverse products of the trans excision-splicing reaction, the trans insertion-splicing reaction is not simply the reverse reaction. The ribozyme recognizes two exogenous substrates through more complex molecular recognition interactions than what has been previously seen in group I intron-derived ribozyme reactions. We give evidence for this new reaction mechanism composed of three steps, with intermediates attached to the ribozyme.

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Agaricus bisporus, le champignon de Paris, est un basidiomycète saprophytenaturellement présent dans la litière de cyprès (Cupressus macrocarpa). Il possède différentsmodes de reproduction. Pour étudier leur rôle dans la dynamique spatio-temporelle et l’évolutionde la diversité génétique au cours du temps, des dispositifs expérimentaux ont été mis en place.Dix souches sauvages d’Agaricus bisporus var. bisporus ont été sélectionnées, à partir de deuxpopulations françaises, sur leurs traits phénotypiques et génotypiques. L’étude de l’évolutionmoléculaire de leurs génomes a montré que, pour le génome mitochondrial, la mobilité desintrons de groupe I apparait comme la principale source de polymorphisme. Des taux desubstitution nucléotidique (nt) faibles ont été observés chez tous les types de séquencesmitochondriales (éléments mobiles, séquences géniques et inter géniques). Cette forteconservation, comparée avec les taux élevés de substitution nt des séquences nucléairessimilaires, contraste avec ce qui est généralement décrit dans l’évolution des séquencesfongiques. Des expériences de croisements entre sporocarpes et mycelia de souches sauvages ontété menées sur du compost, dans une chambre de culture, pour simuler l’implantation d’unepopulation naturelle. Pour les sporocarpes récoltés, les données montrent l’existence d’unphénomène parasexuel de Buller conduisant à des souches hybrides d’A. bisporus dans lachambre de culture et potentiellement dans la nature. Parallèlement, les mycelia de souchessauvages ont été introduits dans deux parcelles expérimentales de cyprès. L’analyse génotypiquedes sporocarpes récoltés la première année d’introduction n’a pas permis de mettre en évidencede souche hybride et les conditions climatiques de la seconde année n’ont pas permis d’obtenir defructification. Les dispositifs et outils mis au point doivent permettre un suivi génétique spatiotemporelde la population sur le long terme. / Agaricus bisporus, the button mushroom, is a saprophytic basidiomycete naturallyfound in cypress litter (Cupressus macrocarpa). It possesses different modes of reproduction. Tostudy their role in the spatio-temporal dynamics and in the evolution of the genetic diversity,experimental systems have been set up. Ten wild strains of Agaricus bisporus var. bisporus havebeen selected, from two french populations, on both phenotypic and genotypic traits. Themolecular evolution of their genomes has shown that, for the mitochondrial genome, group Iintron mobility was the main source of gene polymorphism. Low nucleotide (nt) substitutionrates were found in all types of mitochondrial sequences (mobile elements, genic and intergenicones). This stringent conservation of mitochondrial sequences, when compared with the high ntsubstitution rates of their nuclear counterparts, contrasts to what is widely accepted in fungalsequence evolution. Mating experiments between sporocarps and mycelia of wild strains wereconducted on compost in a room culture, to simulate the implantation of a natural population.Among the collected sporocarps, results indicate the occurrence of a parasexual Bullerphenomenon leading to hybrid strains of A. bisporus in room culture and putatively in the wild.In parallel, mycelia of the wild strains have been introduced in two experimental plots of cypress.Genotypic analysis of the sporocarps collected from these plots in the first year of introduction,failed to evidence a hybrid strain. The climatic conditions of the second year did not allowobtaining fruiting-bodies. The developed experimental systems and tools must allow a followingat the genetic level of the spatio-temporal evolution of the population.

This work presents a survey of blue-stain fungi found in Northwestern Ontario, characterization of a homing endonuclease gene within Grosmannia piceiperda and finally an examination of the introns and homing endonuclease genes found in the large ribosomal subunit gene in species of Ceratocystis; using molecular techniques and phylogenetic analysis, we studied the molecular evolution of these mobile genetic elements. The blue-stain fungi of Northwestern Ontario were identified based on phylogenic analysis of rDNA internal transcribed spacer region sequences. This data was supplemented with morphological characteristics of the fungal cultures. The second project was an examination of a LAGLIDADG homing endonuclease and its IC2 group I intron. This intron is uniquely positioned within the group I intron-encoded rps3 gene of the large subunit ribosomal RNA gene. The final chapter is an investigation of the large subunit ribosomal RNA gene in species of Ceratocystis. The 3’ segment of this gene contains several novel introns and homing endonuclease genes. There is also much diversity between strains despite their close relation on the rDNA internal transcribed spacer region phylogenetic tree. Further, our data also suggest that the single motif LAGLIDADG homing endonuclease of the rDNA mL1923 intron is likely to be an ancestor to other homing endonucleases in the area. The results of these studies demonstrate the role that these elements play in the genetic diversity observed in the blue-stain fungi.

This work presents a survey of blue-stain fungi found in Northwestern Ontario, characterization of a homing endonuclease gene within Grosmannia piceiperda and finally an examination of the introns and homing endonuclease genes found in the large ribosomal subunit gene in species of Ceratocystis; using molecular techniques and phylogenetic analysis, we studied the molecular evolution of these mobile genetic elements. The blue-stain fungi of Northwestern Ontario were identified based on phylogenic analysis of rDNA internal transcribed spacer region sequences. This data was supplemented with morphological characteristics of the fungal cultures. The second project was an examination of a LAGLIDADG homing endonuclease and its IC2 group I intron. This intron is uniquely positioned within the group I intron-encoded rps3 gene of the large subunit ribosomal RNA gene. The final chapter is an investigation of the large subunit ribosomal RNA gene in species of Ceratocystis. The 3’ segment of this gene contains several novel introns and homing endonuclease genes. There is also much diversity between strains despite their close relation on the rDNA internal transcribed spacer region phylogenetic tree. Further, our data also suggest that the single motif LAGLIDADG homing endonuclease of the rDNA mL1923 intron is likely to be an ancestor to other homing endonucleases in the area. The results of these studies demonstrate the role that these elements play in the genetic diversity observed in the blue-stain fungi.

Abstract Species of Lactobacillus (L.) are important starter and probiotic lactic acid bacteria used in the dairy industry. Industrial fermentation processes are prone to phage infections, which can cause severe economic losses. The main objective of this thesis was to examine in more depth the genetic variation and evolution of L. delbrueckii and L. rhamnosus phages. Aspects of interactions and co-evolution of a phage and its host have also been included in this study. In this study, the complete genomic DNA sequences of four Lactobacillus phages were determined and analyzed in detail. Specific phage genes and genetic elements were identified and studied in more depth. The L. delbrueckii phage JCL1032 was found to be a temperate phage which is able to integrate into two distinct genes of L. delbrueckii, but with exceptionally low frequency. The isolated JCL1032-lysogenic bacteria expressed a complex phage resistance against several L. delbrueckii phages. The rarely reported coexistence of phage adsorption resistance and immunity could not be explained by lysogenic conversion. Instead, the spontaneously induced JCL1032 may have provided a selective advantage to adsorption resistant lysogens. The biological activity of two group I introns residing within the terminase large subunit and tape measure genes of the JCL1032 genome (49,433 bp) was demonstrated. The diversification of L. delbrueckii phages is mainly due to insertions, deletions and recombination, as was demonstrated by comparative analyses of the LL-Ku and c5 genomes of 31,080 bp and 31,841 bp, respectively. Interestingly, both phages have possible autonomous transcription units of genes within their genomes. It seemed that evolution of the 36,366-bp genome of the L. casei phage Lc-Nu has been fuelled by deletions as well. The lytic phage Lc-Nu has an imperfect lysogeny module and the phage is genetically closely related to L. casei prophages. This clearly demonstrated that Lc-Nu has a recent temperate origin. This study provides genetic tools, genes, and regulatory elements for biotechnological applications and for developing starter strains with enhanced phage resistance properties. / Tiivistelmä Hapatteina ja probiootteina käytetyt Lactobacillus-maitohappobakteerit (L. ) ovat merkittävässä asemassa meijeriteollisuudessa. Teolliset käymisprosessit ovat alttiita faagi-infektioille, jotka voivat aiheuttaa tuotantolaitoksille huomattavia taloudellisia tappioita. Tämän tutkimuksen päätavoitteena oli syventää tietoa L. delbrueckii ja L. rhamnosus -bakteereita infektoivien faagien geneettisestä muuntelusta ja evoluutiosta. Tutkimuksessa käsitellään myös faagin ja isäntäbakteerin välistä vuorovaikutusta sekä yhteisevoluutiota. Tutkimuksessa määritettiin neljän Lactobacillus-faagin genominen DNA-sekvenssi, identifioitiin faagigeenejä ja muita geneettisiä elementtejä sekä tutkittiin niiden toimintaa. L. delbrueckiin JCL1032-faagi osoittautui tutkimuksessa temperaatiksi. JCL1032-genomi voi integroitua kahteen eri geeniin isäntäbakteerin kromosomissa, joskin lysogeniafrekvenssi on hyvin alhainen. Tutkimuksessa eristetyt JCL1032-lysogeeniset bakteerikannat olivat resistenttejä useille Lactobacillus-faageille. Osassa lysogeenisia bakteereita resistenssi ilmeni jo faagin adsorptiovaiheessa. Vastaavanlainen ilmiö on kuvattu vain harvoin aiemmin. Havaittua kompleksista resistenssiä ei voitu selittää lysogeenisella konversiolla. Sen sijaan ilmiön taustalla voi olla JCL1032-profaagien spontaani indusoituminen bakteerin kromosomista, mikä voi antaa valintaetua adsorptioresistenteille lysogeenisille bakteereille. JCL1032-genomissa (49 433 emäsparia) osoitettiin olevan kaksi biologisesti aktiivista intronia terminaasin suurta alayksikköa ja hännän mittaproteiinia koodaavissa geeneissä. LL Ku- ja c5-faagien genomien (31 080 ja 31 841 emäsparia) vertailu osoitti L. delbrueckii -faagien evoluution olevan pääasiassa seurausta insertioista, deleetioista ja rekombinaatiosta. Kummassakin genomissa oli mahdollisesti päällekkäisiä ja itsenäisesti transkriptoituvia geenialueita. Deleetiot ovat muokanneet myös L. casein lyyttisen Lc- Nu-faagin genomia (36 466 emäsparia). Faagin lysogeniamoduuli sisälsi vain osan lysogeeniseen elinkiertoon tarvittavista geeneistä. Lc-Nu on geneettisesti läheistä sukua L. casei -profaageille, mikä myös viittaa siihen, että Lc-Nu on kehittynyt temperaatista faagista. Tutkimustuloksia faagien geeneistä ja säätelyelementeistä voidaan hyödyntää hapatebakteerien faagiresistenssiominaisuuksien kehittämisessä sekä erilaisissa bioteknologisissa sovelluksissa.

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