Brain metastasis (BM), the most common tumor of the central nervous system, occurs in 20-36% of primary cancers. In particular, 20-40% of patients with non-small cell lung cancer (NSCLC) develop brain metastases, with a dismal survival of approximately 4-11 weeks without treatment, and 16 months with treatment. This highlights a large unmet need to develop novel targeted therapies for the treatment of lung-to-brain metastases (LBM). Genomic interrogation of LBM using CRISPR technology can inform preventative therapies targeting genetic vulnerabilities in both primary and metastatic tumors. Loss-of-function studies present limitations in metastasis research, as knocking out genes essential for survival in the primary tumor cells can thwart the metastatic cascade prematurely. However, transcriptional overexpression of genes using CRISPR activation (CRISPRa) has the potential for overcoming dependencies of gene essentiality. In this thesis, we created and utilized an in vivo genome-wide CRISPRa screening platform to identify novel genes, that when overexpressed, drive LBM. We have developed a patient-derived orthotopic murine xenograft model of LBM using a patient-derived NSCLC cell line (termed CRUK cells) from the Swanton Lab TRACERx study. We introduced a human genome-wide CRISPRa single guide RNA (sgRNA) library into non-metastatic and pro-metastatic lung cancer CRUK cells to achieve 500X representation of each sgRNA in the activation library. We then injected the cells into the lungs of immunocompromised mice and tracked lung tumor development and BM formation. Upon sequencing primary lung tumors and subsequent BM, we will identify enriched sgRNAs which may represent novel drivers of primary lung tumor formation and LBM. To the best of our knowledge, this study is the first in vivo genome-wide CRISPR activation screen using patient-derived NSCLC cells to help elucidate drivers of LBM. This work serves to provide a framework to gain a deeper understanding of the regulators of BM formation which will hopefully lead to targeted drug discovery that will ultimately be used in clinical trials to help eradicate brain metastasis in NSCLC patients. / Thesis / Master of Science (MSc) / Brain metastasis, or the spread of a primary cancer from another organ to the brain, is the most common adult brain tumor. Brain metastases can arise after the treatment of primary tumors and are only detected in the clinic at a highly malignant stage. Current treatments for brain metastasis consist of surgical removal and palliative chemoradiotherapy, which fail to fully eliminate the brain tumor. Over 20% of cancer patients develop brain metastases, with lung, breast, and skin cancers leading as the top three sources of metastasis. In particular, 40% of patients with non-small cell lung cancer develop brain metastasis, with survival of only 4-11 weeks once diagnosed without treatment, and 16 months with treatment. As systemic therapies for the treatment of non-small cell lung cancer are becoming increasingly effective at controlling primary disease, patients are ironically succumbing to their brain tumors. This highlights a large unmet need to develop novel targeted therapies for the treatment of lung-to-brain metastases (LBM). Functional genomic tools provide the opportunity to investigate the genetic underpinnings of LBM. With the advent of gene editing technologies, we are able to overexpress various genes and observe the impact genetic perturbations have on tumor initiation, growth, and metastasis. In this thesis, we devised a pre-clinical animal model of LBM that could be used to study genetic drivers of LBM using a gene overexpression tool such that one gene per tumor cell gets activated. We are then able to model the disease trajectory from a lung tumor to brain metastasis development using patient samples in our animal model and identify genes that, upon overexpression, drive LBM. This platform will lead to potential therapeutic targets to prevent the formation of LBM and prolong the survival of patients with non-small cell lung cancer.

<p> This thesis focused on the development of comprehensive, rapid and simple methodologies for the analysis of fatty acids by gas chromatography mass spectrometry (GC/MS) and intact lipids by electrospray ionization tandem mass spectrometry (ESIIMS/MS). The methodologies were applied as a tool for functional genomics in the soil bacterium Sinorhizobium meliloti. The effects of inorganic phosphate (Pi)-starvation and acidity on lipid composition were studied. </p> <p> A micro-scale, one-vial method for the analysis of fatty acids as their fatty acid methyl esters by GC/MS was developed. The method required small sample sizes, involved minimum handling and avoided tedious extraction steps, which increased sample throughput. A series of quality controls were included to measure losses due to handling, derivatization efficiencies and the extent of side reactions. The method was suitable for the analysis of sensitive bacterial fatty acids such as cyclopropane fatty acids. </p> <p> A shotgun lipidomics approach was developed for the analysis of intact lipids by ESIIMS/MS. Fatty acid distributions were obtained for eight lipid classes and up to 58 individual lipids were identified in crude lipid extracts without sample cleanup or chromatography. For the first time, fatty acid distributions were provided for non-phosphorus containing lipids using shotgun lipidomics. Fatty acid distributions within lipid classes suggested that phospholipids and 1,2-diacylglyceryl-3-O-4'-(N,N,N-trimethyl)-homoserine lipids (TMHSs) were both synthesized from phosphatidic acid while sulfoquinovosyldiacylglycerol (SLs) had a different biosynthetic origin. </p> <p> The methodologies were applied to study knockout mutants of five genes thought to participate in lipid metabolism in S. meliloti. It was demonstrated that: (1) cfa2 gene coded for the main cyclopropane fatty acyl synthase; (2) the plsC gene coded for a fatty acy 1 transferase specific for C 16 fatty acids in the sn-2 position of phospholipids; (3) a metabolic phenotype was revealed for knockout mutants of dme and tme genes (DME and TME, malic enzymes) when succinate was the carbon source. </p> / Thesis / Doctor of Philosophy (PhD)

No description available.

Glioblastoma (GBM) remains the most aggressive and prevalent malignant primary brain tumor in adults. Unchanged since 2005, standard of care (SoC) consists of surgical resection, followed by radiation therapy (RT) with concurrent and adjuvant chemotherapy with temozolomide (TMZ). Despite these therapeutic efforts, patients succumb to recurrent disease with a median overall survival of 14.6 months and a five-year survival rate of 5.5-6.8%. Therapeutic failure is largely explained by ITH and the presence of treatment-resistant GBM stem-like cells (GSCs). Given the lack of understanding of recurrent GBM and absence of second line therapies patients, I hypothesize that genome-scale functional genetic interrogation will unravel recurrent GBM-specific tumor biology and inform development of novel therapeutics. First, I compared primary and recurrent GBM at the genetic, transcriptomic, proteomic and functional genetic levels. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Mechanistically, genetic perturbation and a small molecule inhibitor of PTP4A2 repress axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and exploit a genetic dependency on ROBO signaling. Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition. Given the genetic dependency on ROBO signaling and enrichment of ROBO1 expression in GBM tissues, I undertook a campaign to evaluate ROBO1 as a therapeutic target in recurrent GBM and develop anti-ROBO1 chimeric antigen receptor T (CAR-T) cells using camelid single-domain antibodies targeting human ROBO1. I optimized the design of anti-ROBO1 CAR-T cells and tested the anti-tumor activity of these modalities in in vitro using patient-derived recurrent GBM lines and orthotopic patient-derived xenograft models. I present data to expand the repertoire of GBM-enriched antigens suitable for effective CAR-T cell therapy. Given that resistance to SoC and disease relapse are inevitable for GBM patients, pre-clinical and clinical advancement of immunotherapeutic modalities, combined with recent insights into the tumor immune microenvironment, are poised to improve clinical outcomes for this patient population. / Thesis / Doctor of Philosophy (PhD) / Glioblastoma remains the most lethal and prevalent primary brain tumor in adults. Standard of care for patients remains unchanged since 2005, consisting of surgery to remove visible tumor at diagnosis (primary tumor), followed by radiation therapy and chemotherapy to treat remaining tumor cells. Despite these therapeutic efforts, tumor relapse (recurrent tumor) is inevitable with no standardized second-line therapy. Patients succumb to recurrent disease with a median overall survival of 14.6 months and only 5.5-6.8% of patients survive five years post diagnosis. Therapy failure and tumor relapse are explained by immense diversity among tumor cells at the DNA and protein levels, giving rise to a subset of tumor cells with abilities to resist therapy and seed the recurrent tumor. Previous studies have presented evolution of tumor cells through therapy, with recurrent tumor cells harboring novel changes at the DNA and protein levels. However, the impact of these changes on tumor cell function has not been evaluated. In this thesis, we developed and applied a genetic screening technique to determine the functional role of thousands of genes in primary and recurrent tumor cells from the same patient. This analysis revealed numerous genes that exhibit differential effects on survival of primary and recurrent tumor cells, including genes that drive recurrent tumor cell growth but are dispensable in primary tumor cells. Functional remodeling of these genes and pathways revealed a new functional role of multiple proteins belonging to a process called axonal guidance in recurrent tumor cells. To evaluate the therapeutic potential of these findings, we deeply interrogated the mechanism by which axonal guidance drives recurrent tumor cells and targeted crucial molecular players using chemical and immunological therapies. Using models that predict clinical effectiveness, we engineered and tested a novel therapy that redirects immune cells to target recurrent tumor cells driven by dysfunctional axonal guidance activity. The goal of this thesis was to discover the functional differences between primary and recurrent tumor cells, thereby leveraging this information to engineer candidate therapies for treatment of recurrent glioblastoma.

Much interest has recently accumulated of the role of adult stem cells in both normal tissue homeostasis and carcinogenesis. Whereas normal and cancerous mammary epithelial stem cells have been identified and isolated from bulk primary tissue, little remains known about their regulation in vivo. Here we describe the molecular profile of mammary epithelial stem cells cultured in vitro and that of their tumourigenic counterparts, breast cancer stem cells. Our studies of gene transcription reveal potential mechanisms that may cooperate in the regulation of normal and cancer stem cells in vitro, and may also reflect their in vivo behaviour. These data bear consequences for the design of novel breast cancer therapeutics, as cancer stem cells are thought to resist conventional treatments and persist thereafter, causing disease relapse and seeding metastases. To address this issue we have devised a functional genomics approach to screen for novel biomarkers and therapeutic targets of breast cancer modeled in vitro; this culture system is centered on bona fide stem cells and may therefore offer improved relevance to human disease when compared with breast cancer cell lines. / Thesis / Master of Science (MS)

Leptosphaeria maculans est un Dothideomycète phytopathogène capable d’alterner des modes de vie saprophyte, hemibiotrophe endophyte et nécrotrophe durant son cycle infectieux sur le colza. Afin de comprendre cette plasticité infectieuse, une mutagenèse aléatoire à grande échelle par ATMT a permis de générer une collection de 5000 transformants. L’objectif principal de cette thèse était d’exploiter cette collection en prenant avantage de la disponibilité d’outils de génomique, pour, d’une part, comprendre les mécanismes d’intégration de l’ADN-T dans le génome, et d’autre part, identifier des déterminants du pouvoir pathogène ciblés par l’ADN-T dans des mutants affectés dans leur capacité infectieuse. Une analyse systématique de 318 pieds d’insertion a été réalisée dans le but de d’évaluer les caractéristiques de l’insertion de l’ADN-T. Ce dernier est fréquemment inséré dans les régions actives riches en gènes, et favorise des gènes impliqués dans des processus biologiques indicatifs d’une conidie germante. Un biais marqué des insertions en faveur des régions régulatrices est observé ainsi qu’une corrélation entre la densité des insertions et le skew CG près du site d’initiation de la transcription. Ces observations sont cohérentes avec le modèle de ciblage intranucléaire de l’ADN-T. Enfin, l’existence de micro-homologies entre le site de pré-insertion et la bordure gauche de l’ADN-T indique une intégration par voie de recombinaison homologue (HR) et/ou microhomologue (MMEJ). Une approche multicritère a été utilisée pour caractériser cette collection. Un test d’inoculation a permis d’identifier 166 mutants altérés dans leur pouvoir pathogène. La validation génétique de la co-ségrégation entre le phénotype altéré et la présence de l’ADN-T indique que 44% des mutants montrant un déterminisme monogénique de l’altération sont effectivement étiquetés. Parmi les mutants altérés, 35 ont été analysé pour : (i) le phénotype de croissance en conditions usuelles de culture et en réponse aux stress oxydatif, UV et nutritif, (ii) le lien entre altération de la germination et pouvoir pathogène. Les gènes affectés par l’intégration de l’ADN-T, ont été identifié et analysé dans la souche sauvage à l’aide de données de transcriptomique. Ces cribles ont permis de décomposer le phénotype d’altération in planta en plusieurs phénotypes in vitro. Le plus fréquemment, les mutants ne sont pas altérés dans leur in vitro croissance, mais la plupart sont sensibles aux ROS. Les analyses d’expression au cours de l’infection indiquent que les gènes codant pour des effecteurs et ceux impliqués dans la détoxification des ROS, ont des dynamiques d’expression inverses et bi-phasiques, en lien avec le style de vie hemibiotrophe de L. maculans. Les 42 gènes ciblés par l’ADN-T dans ces mutants ont été identifiés et leur fonction putative disséquée par bioinformatique et transcriptomique. Au final, nous avons identifié six mutants d’intérêt pour une caractérisation fonctionnelle. Deux de ceux-ci deux ont atteint un niveau de caractérisation suffisant pour l’émission d’une hypothèse de travail. Dans le mutant µ1165, l’ADN-T cible un gène codant pour une protéine ribosomale S17 (LmRPS17), dont la régulation dépendrait de la voie de signalisation du complex TORC1. Nos résultats préliminaires suggèrent, d’une part, que TORC1 est une cible potentielle de LmRPS17 et, d’autre part, que la phase biotrophe de l’infection chez L. maculans est probablement régulée par TORC1 via l’enzyme de détoxication des ROS SOD2. Dans le mutant µ444, l’ADN-T cible un gène codant pour un élément rétroviral putatif LmHYP1, largement conservé parmi les ascomycètes. Nos résultats suggèrent que LmHYP1 interviendrait dans la régulation de gènes impliqués dans le pouvoir pathogène, via la production de petits ARN interférents issus hypothétiquement du clivage de son ARN messager par la machinerie de défense anti-rétrovirale. La validation de ces deux hypothèses est en cours au laboratoire. / The Dothideomycete phytopathogen Leptosphaeria maculans is capable to alternate saprophytic, hemibiotrophic, endophytic and necrotrophic life styles during a single infectious cycle on its host plant, Brassica napus. However, little is known about the determinants of such plasticity. As part of a large-scale insertional mutagenesis project aiming at the discovery of pathogenicity factors a collection 5000 transformants has been generated by ATMT. The main objective of my PhD was to take advantage of “omics” to extract biological value from L. maculans mutants collection for a better understanding of T-DNA integration features and further identification of pathogenesis determinants in this fungus. A systematic analysis of 318 T-DNA tags was performed in a large collection of transformants in order to evaluate the features of T-DNA integration in its particular TE-rich compartmentalized genome. The T-DNA integration was mainly targeted to gene-rich, transcriptionally active regions, and favored biological processes that are consistent with the physiological status of a germinating conidia. In addition, T-DNA integration was strongly biased towards regulatory regions, and mainly promoters. Consistently with the T-DNA intranuclear targeting model, the density of T-DNA insertion correlated with CG skew near the transcription initiation site. The existence of microhomologies between promoter sequences and the T-DNA LB flanking sequence was also consistent with T-DNA integration to host DNA mediated by homologous recombination and/or the microhomology-mediated end joining pathways. Based on this data, a multi-criteria approach was used to draw the more possible information from this collection by identifying all 166 mutants reliably affected in pathogenicity, and expanding the genetic analysis. Considering single-gene segregation of the pathogenicity phenotype, our data indicate a 44% ratio of actual tagging. In parallel, for a subset of 35 isolates of the collection, we (i) described growth patterns in regular culture conditions or in response to oxidative, UV or starvation stresses, (ii) evaluated the link between altered germination and pathogenicity, (iii) identified and annotated the genes putatively affected by the T-DNA integration, and (iv) analyzed kinetics of expression of these genes in the WT isolate using available microarrays. Our results showed that pathogenicity alteration phenotype could be broken down into a pattern of phenotypes in vitro including growth, germination defect and susceptibilities to oxidative burst, starvation and UV stresses. Our results showed that most of these mutants were not altered in axenic growth but showed enhanced sensitivity to reactive oxygen species (ROS). Furthermore, our results showed that effectors and ROS scavengers have inverted dynamics during plant infection, consistently with the biphasic hemibiotrophic growth of L. maculans. Also, 42 genes targeted by the T-DNA in these mutants were recovered and dissected. This catalogue allowed us to identify a series of promising mutants for further functional studies. Based on this screening, six mutants were subjected to further analyses but only two reached sufficient advance for hypothesis building. In mutant µ1165, the T-DNA targeted a ribosomal protein S17 (LmRPS17), a downstream component of target of rapamycin complex 1 (TORC1) pathway. Our preliminary results suggested that TORC1 is a potential a target of LmRPS17. Also, biotrophic growth is probably tuned by TORC1 via Super oxide dismutase 2 (SOD2). In mutant µ444, the T-DNA targeted a retroviral-like element LmHyp1 widely conserved among ascomycetes. Our results suggest that LmHyp1 probably acts as a regulatory element during plant infection as its cleavage by the antiretroviral defences is hypothesized to generate siRNAs that silences distant genes. Work on these mutants is in progress.

No description available.

Le but de cette thèse est de préciser les mécanismes moléculaires agissant en aval du facteur de transcription CROWN ROOT LESS 1 (CRL1) qui régule la formation des racines coronaires (RC). Nous avons pu identifier dans un premier temps une nouvelle séquence d’ADN reconnue par CRL1 nommé CRL1-box différente de la LBD-box qui était le seul motif cis-régulateur précédemment décrit pour la famille des facteurs de transcription (FT) de type LATERAL ORGAN BOUNDARIES DOMAIN (LBD). Nous avons ensuite identifié un groupe de gènes régulés par CRL1, et avons montré l’implication de deux d’entre eux, OsROP et OsbHLH044, dans le développement des RC. OsbHLH044 est un facteur de transcription répresseur et semble être aussi impliqué dans la sénescence cellulaire ainsi que la réponse aux stress. Enfin, nous avons mis en évidence une cascade de régulation liant positivement CRL1 avec QUIESCENT-CENTER-SPECIFIC HOMEOBOX (QHB) un gène impliqué dans la différenciation et le maintien du centre quiescent via le facteur de transcription OsHOX14. En addition nous avons mis en évidence une boucle de rétroaction négative de QHB sur ses activateurs CRL1 et OsHOX14, qui pourrait être impliquée dans la structuration du primordia de racine coronaire. / The aim of this thesis is to specify the molecular mechanisms acting downstream of the CROWN ROOT LESS 1 transcription factor (CRL1) that regulates coronary root (CR) formation. We were able to identify at first a new CRL1 recognized DNA sequence named CRL1-box different from the LBD-box which was the only cis-regulatory motif previously described for the LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factor (TF) family. We then identified a group of genes regulated by CRL1, and showed the involvement of two of them, OsROP and OsbHLH044, in the development of CR. OsbHLH044 is a repressive transcription factor and appears to be also involved in cell senescence as well as stress response. Finally, we demonstrated a regulatory cascade linking CRL1 with QUIESCENT-CENTER-SPECIFICHOMEOBOX (QHB), a gene involved in the differentiation and maintenance of the quiescent center, via the OsHOX14 transcription factor. In addition we have demonstrated a negative feedback loop of QHB on its activators CRL1 and OsHOX14, which could be involved in structuring the coronary root primordia

xiii, 145 p. : ill. (some col.) / The goal of functional genomics is to elucidate the relationship between an organism's genotype and phenotype. A key characteristic of functional genomics is the use of genome-wide approaches as opposed to more traditional single-gene approaches. Genome-wide expression profiling is used to investigate the dynamic properties of transcriptomes, provides insights into how biological functions are encoded in genomes, and is an important technique in functional genomics. This dissertation describes the use of genome-wide expression profiling and other functional genomics techniques to address a variety of biological questions related to development and disease of the nervous system. Our results reveal novel and important insights into nervous system development and disease and demonstrate the power of functional genomics approaches for the study of nervous system biology. This dissertation also describes a novel technique called TUtagging that facilitates cell type-specific RNA isolation from intact complex tissues. The isolation of RNA from specific cell types within a complex tissue is a major limiting factor in the application of genome-wide expression profiling, and TU-tagging can be used to address a wide array of interesting and important biological questions. This dissertation includes previously published and unpublished co-authored material. / Committee in charge: Dr. John Postlethwait， Chair； Dr. Chris Doe， Advisor； Dr. Bruce Bowerman， Member； Dr. Patrick Phillips， Member； Dr. Tom Stevens， Outside Member

Os micoplasmas são os menores microrganismos capazes de autoreplicação conhecidos na natureza, responsáveis por uma série de doenças no homem e nos animais, infectando ainda plantas e insetos. Constituem um grande grupo de bactérias, ordenadas em diferentes gêneros na classe Mollicutes, cuja principal característica em comum, além do genoma reduzido, é a ausência de parede celular. Mycoplasma mycoïdes subsp. mycoïdes SC, responsável pela Pleuropneumonia Contagiosa Bovina, foi o primeiro microrganismo desta classe de bactérias a ser identificado. Esta é uma doença bastante grave, com altas taxas de morbidade e mortalidade. A variedade Mycoplasma mycoïdes subsp. mycoïdes LC é responsável principalmente por casos de Pleuropneumonia Contagiosa Caprina, mastite no gado bovino, e ainda artrite em ovinos e caprinos em menor extensão. M. capricolum é um patógeno caprino, responsável principalmente por casos de artrite com grande importância econômica na medicina veterinária. M. pulmonis é um patógeno de roedores, considerado como o melhor modelo experimental para o estudo das micoplasmoses respiratórias. M. genitalium, o menor microrganismo conhecido capaz de se autoreplicar, é um patógeno humano responsável por casos de uretrite não gonocócica, cujo seqüenciamento completo do cromossomo tornou-se um marco na era da genômica. O estudo funcional do genoma destes micoplasmas, para a compreensão de sua biologia e patogenicidade, requer o desenvolvimento de ferramentas genéticas eficientes. No presente trabalho, análises in silico das seqüências na região das prováveis origens de replicação cromossômica (oriC) destes micoplasmas demonstraram a existência de possíveis DnaA boxes localizados em torno do gene dnaA. Estas regiões oriC foram caracterizadas funcionalmente após sua clonagem em vetores artificiais e a transformação dos micoplasmas com os plasmídeos recombinantes resultantes. O plasmídeo pMPO1, contendo a região oriC de M. pulmonis, sofreu integração no cromossomo do micoplasma por recombinação homóloga após poucas passagens in vitro. A redução desta oriC para o fragmento contendo somente os DnaA boxes localizados nas estremidades 5´ou 3´do gene dnaA não foi capaz de produzir plasmídeos replicativos em M. pulmonis, exceto quando estes dois fragmentos foram clonados no mesmo vetor, espaçados pelo determinante de resistência à tetraciclina tetM. Um fragmento interno do gene da hemolisina A (hlyA) de M. pulmonis foi clonado nestes plasmídeos oriC, e os vetores resultantes foram utilizados para transformar o micoplasma. A integração destes vetores por um crossing-over com o gene hlyA, causando a sua dirupção, foi documentada. Deste modo, estes plasmídeos oriC podem vir a se tornar ferramentas genéticas valiosas para o estudo do papel de genes específicos, notadamente aqueles potencialmente envolvidos na patogênese. / Mycoplasmas are the smallest microorganisms capable of self replication known to date, responsible for many diseases in man and animals, infecting also plants and insects. They constitute a large group of bacteria, classified in different genera in the class Mollicutes, which main common characteristic, besides the small genome, is the absence of a cell wall. Mycoplasma mycoïdes subsp. mycoïdes SC, responsible for the Bovine Contagious Pleuropneumonia, was the first microorganism of this class of bacteria to be identified. That is a quite severe disease, with high morbidity and mortality rates. Mycoplasma mycoïdes subsp. mycoïdes LC is responsible mainly for cases of Caprine Contagious Pleuropneumonia, mastitis in cattle, and also arthritis in goats and sheep in less extension. M. capricolum is a pathogen of goats, responsible mainly by cases of arthritis with large economic impact in veterinary medicine. M. pulmonis is a rodent pathogen, considered to be the best experimental model for studying respiratory mycoplasmoses. M. genitalium, the smallest microorganism capable of self replication, is an human pathogen responsible for cases of non gonococcal urethritis, which complete chromosome sequencing has become a benchmark in the era of genomics. Functional studies of these mycoplasma genomes, for comprehension of their biology and pathogenicity, requires the development of efficient genetic tools. In the present work, in silico analysis of sequences of the putative origin of chromosome replication (oriC) region of these mycoplasmas demonstrates the existence of putative DnaA boxes located around the dnaA gene. These oriC regions were functionally characterized after cloning into artificial vectors and transformation of mycoplasmas with the resulting recombinant plasmids. The plasmid pMPO1, which contains the M. pulmonis oriC region, has integrated into the mycoplasma chromosome by homologous recombination after a few in vitro passages. Reduction of this oriC to the fragment containing only the DnaA boxes located upstream or downstream the dnaA gene could not produce plasmids able to replicate in M. pulmonis, except when these two fragments were cloned in the same vector, spaced by tetracycline resistance gene tetM. An internal fragment of the M. pulmonis hemolysine A gene (hlyA) was cloned into these oriC plasmids, and the resulting vectors were used to transform the mycoplasma. Integration of these disruption vectors by one crossing-over with the hlyA gene could be documented. Therefore, these oriC plasmids may become valuable genetic tools for studying the role of specific genes of mycoplasmas, specially those potentially involved in pathogenesis.