mark laflamme | Fisheries and Oceans Canada (original) (raw)
Papers by mark laflamme
The FASEB Journal, 2007
ABSTRACT 5-lipoxygenase (5-LO) catalyzes the initial reactions leading to the conversion of arach... more ABSTRACT 5-lipoxygenase (5-LO) catalyzes the initial reactions leading to the conversion of arachidonic acid into a number of biologically important mediators of inflammation. We seek to better understand the control of the biosynthesis of 5-LO products which is linked to inflammatory diseases such as asthma and atherosclerosis. In this study we used the human acute lymphocytic leukemia and Burkitt lymphoma cell lines REH and Raji, respectively, to investigate whether alternative variants of 5-LO mRNA are produced. Using reverse-transcription of extracted mRNA coupled with PCR, followed by molecular cloning and sequencing experiments, we have identified 4 different variants of 5-LO mRNA in these cell lines including the full length mRNA containing all 14 exons with the expected splicing sites. Amongst the alternative isoforms, one resulting from the exclusion of exon 13 was identified in both cell lines and was present in approximately 30% of the generated clones. This exclusion of exon 13 does not induce a reading frame shift but would result in a translation product missing 57 amino acids near the carboxyl terminal. Experiments are underway to determine whether the translation products of these variant isoforms are expressed and have an impact on the capacity of cells to synthesize 5-LO products. (Supported by the Canadian Institutes of Health Research and the Canada Research Chairs Program)
Journal of Fish Diseases
The infectious salmon anaemia virus (ISAV) is the causative agent of infectious salmon anaemia (I... more The infectious salmon anaemia virus (ISAV) is the causative agent of infectious salmon anaemia (ISA), an important disease of farmed Atlantic salmon (Salmo salar L.) that has resulted in a regulatory control programme for Canada and the development of international standards by the World Organisation for Animal Health (OIE).
Molecular ecology resources, Jan 21, 2016
The increasing use of high-throughput sequencing platforms has made the isolation of pure, high m... more The increasing use of high-throughput sequencing platforms has made the isolation of pure, high molecular weight DNA a primary concern for studies of a diverse range of organisms. Purification of DNA remains a significant challenge in many tissue and sample types due to various organic and inorganic molecules that coprecipitate with nucleic acids. Molluscs, for example, contain high concentrations of polysaccharides which often coprecipitate with DNA and can inhibit downstream enzymatic reactions. We modified a low-salt CTAB (MoLSC) extraction protocol to accommodate contaminant-rich animal tissues and compared this method to a standard CTAB extraction protocol and two commercially available animal tissue DNA extraction kits using oyster adductor muscle. Comparisons of purity and molecular integrity showed that our in-house protocol yielded genomic DNA generally free of contaminants and shearing, whereas the traditional CTAB method and some of the commercial kits yielded DNA unsuita...
Fish & Shellfish Immunology, 2016
The Infectious salmon anemia virus (ISAV) is an important viral disease of farmed Atlantic salmon... more The Infectious salmon anemia virus (ISAV) is an important viral disease of farmed Atlantic salmon (Salmo salar) which has caused severe financial losses for salmon farmers around the world, including Atlantic Canada. It is listed as an OIE notifiable disease and to this day eradication of infected cages remains the current practice in many countries to mitigate the spread of the virus. All strains known to display any level of virulence are characterised by deletions in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) protein compared to ISAV-HPR0 strains and are designated ISAV-HPR∆. In contrast, HPR0 strains are non-virulent and not detected by viral culture. In Atlantic Canada, improved management and husbandry practices resulted in no outbreaks caused by ISAV-HPR∆ strains from 2007 to 2012, with however ISAV-HPR0 strains (European genotype) still being detected. Starting in 2012, new ISAV-HPR∆ strains were responsible for disease outbreaks in fish farms in Nova Scotia, Newfoundland and New-Brunswick. This study was thus designed to characterize virulence associated to three of the newly identified ISAV-HPR∆ strains and investigate gene expression responses in gills of infected fish using RNA-Seq. All three ISAV-HPR∆ strains studied through the use of in vivo challenges resulted in cumulative mortalities below 40 percent and caused the differential expression of up to 1000 genes at the peak of infection compared to non-infected fish. Many genes linked to innate immunity were over-expressed; similar to what had been observed in the head-kidney through previous work. The use of RNA-Seq also enabled us to look at ISAV transcript abundance and showed that segment 8 had the highest abundance of all 8 segments of the genome. This information has given us great insight on how ISAV interacts with his host during the course of an infection.
Oncotarget, Jan 5, 2017
Pax-5, an essential transcription factor for B lymphocyte development, has been linked with the d... more Pax-5, an essential transcription factor for B lymphocyte development, has been linked with the development and progression of lymphoid cancers and carcinoma. In contrast to B-cell cancer lesions, the specific expression signatures and roles of Pax-5 in breast cancer progression are relatively unknown. In the present study, we set out to profile Pax-5 expression in mammary tissues and elucidate the cellular and molecular roles of Pax-5 in breast cancer processes. Using immunohistology on mammary tissue arrays, Pax-5 was detected in a total of 298/306 (97.6%) samples tested. Interestingly, our studies reveal that Pax-5 inhibits aggressive features and confers anti-proliferative effects in breast carcinoma cells in contrast to its oncogenic properties in B cell cancers. More precisely, Pax-5 suppressed breast cancer cell migration, invasion and tumor spheroid formation while concomitantly promoting cell adhesion properties. We also observed that Pax-5 inhibited and reversed breast can...
Journal of Phycology, 2009
Oligonucleotides, 2008
Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosp... more Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosphoramidite-method DNA synthesis are desirable for many genomics and proteomics applications. In this communication, we present a method for the deprotection of a range of N-acyl deoxyribonucleosides (T, dA Bz , dC Bz , dC Ac , dG ibu , dG PAC ) and synthetic oligodeoxyribonucleotides, ranging in length from 5-mer to 50-mer. Oligodeoxyribonucleotides were synthesized using standard amide protecting groups (dA Bz , dC Bz , dG ibu ) and phosphoramidite chemistry on cis-diol solid phase support. This deprotection method utilizes 29% aqueous ammonia solution at 170°C for 5 minutes under monomode microwave irradiation at a 20-nmole reaction scale. Reaction products were analyzed by TLC, RP-HPLC, CE, ESI-MS, real-time PCR, agarose gel electrophoresis, and by DNA uracil glycosylase (UDG) and phosphodiesterase I (PDE) enzymatic digestions.
Bioinformatics, 2004
Motivation: In the interpretation of gene expression data from a group of microarray experiments ... more Motivation: In the interpretation of gene expression data from a group of microarray experiments that include samples from either different patients or conditions, special consideration must be given to the pleiotropic and epistatic roles of genes, as observed in the variation of gene co-expression patterns.
Journal of Biological Chemistry, 2004
The transcription factor Pax-5 occupies a central role in B cell differentiation and has been imp... more The transcription factor Pax-5 occupies a central role in B cell differentiation and has been implicated in the development of B cell lymphoma. The transcriptional activation function of Pax-5 requires an intact N-terminal DNA-binding domain and is strongly influenced by the C-terminal transactivation domain. We report the identification and characterization of five human Pax-5 isoforms, which occur through the alternative splicing of exons that encode for the C-terminal transactivation domain. These isoforms arise from the inclusion or exclusion of exon 7, exon 8, and/or exon 9. Three of the Pax-5 isoforms generate novel protein sequences rich in proline, serine, and threonine amino acids that are the hallmarks of transactivation domains. The Pax-5 isoforms are expressed in peripheral blood mononuclear cells, cancerous and non-cancerous B cell lines, as well as in primary B cell lymphoma tissue. Electrophoretic mobility shift assays demonstrate that the isoforms possess specific DNA binding activity and recognize the PAX-5 consensus binding sites. In reporter assays using the CD19 promoter, the transactivation properties of the various isoforms were significantly influenced by the changes in the C-terminal protein sequence. Finally, we demonstrate, for the first time, that human Pax-5 isoform expression is modulated by specific signaling pathways in B lymphocytes.
Oligonucleotides, 2008
Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosp... more Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosphoramidite-method DNA synthesis are desirable for many genomics and proteomics applications. In this communication, we present a method for the deprotection of a range of N-acyl deoxyribonucleosides (T, dA Bz , dC Bz , dC Ac , dG ibu , dG PAC ) and synthetic oligodeoxyribonucleotides, ranging in length from 5-mer to 50-mer. Oligodeoxyribonucleotides were synthesized using standard amide protecting groups (dA Bz , dC Bz , dG ibu ) and phosphoramidite chemistry on cis-diol solid phase support. This deprotection method utilizes 29% aqueous ammonia solution at 170°C for 5 minutes under monomode microwave irradiation at a 20-nmole reaction scale. Reaction products were analyzed by TLC, RP-HPLC, CE, ESI-MS, real-time PCR, agarose gel electrophoresis, and by DNA uracil glycosylase (UDG) and phosphodiesterase I (PDE) enzymatic digestions.
Pax-5 is an important contributor to B cell development. Several studies have shown that the dere... more Pax-5 is an important contributor to B cell development. Several studies have shown that the deregulated expression of Pax-5 is implicated in onset of different types of cancers. Previously, we have observed that Pax-5 expression in B-cells increases dramatically upon estrogen stimulation, and that Pax-5 is expressed in breast cancer cell lines while it is undetectable in normal mammary cell lines. Based on this evidence, we hypothesis that Pax-5 may exercise a key role in mammary oncogenesis and that this abnormal function may be modulated by estrogen. In order to shed light on these initial findings, using quantitative real-time RT-PCR we have characterized the Pax-5 expression profiles in several mammary cell lines following stimulation with estrogen and Tamoxifen. We have found that in each case, Pax-5 expression is differentially modulated when comparing treated and untreated cells. Further to this, we are using oligonucleotide microarrays to identify the gene expression pathwa...
The transcription factor Pax5 possesses oncogenic properties and is often deregulated in many sub... more The transcription factor Pax5 possesses oncogenic properties and is often deregulated in many subsets of B-cell lymphomas and leukemias. Furthermore, the Pax5 transcript is known to be alternatively spliced, producing a wide variety of isoforms. It has been recently suggested that deregulation of Pax5 isoforms may lead to these B-cell malignancies. Since aberrant splicing is linked to human diseases, we hypothesized that the deregulation of Pax5 isoforms may result from a defective splicing mechanism caused by mutations in splice sites. Therefore, we have attempted to establish a link between the pattern of expressed Pax5 isoforms in B-cell chronic lymphocytic leukemia (B-CLL) patients and mutations in splice sites. These experiments were performed using PCR amplification of Pax5 in B-CLL patients in conjunction with immunodetection of the protein isoforms by Western blot. Sequencing of PCR products is then used to identify mutations. Our results indicate that full length Pax5 is pr...
Pax-5 is a key regulator of B lymphocyte development, and is known to interact directly or indire... more Pax-5 is a key regulator of B lymphocyte development, and is known to interact directly or indirectly with a large number of protein partners. In order to identify proteins that interact directly with Pax-5, a yeast-two hybrid assay was performed using Pax-5 as bait. A novel interaction between Pax-5 and human nuclear ribonucleoprotein A1 (hnRNP A1) was identified. As hnRNP A1 is a protein involved in RNA processing and could influence Pax-5- mediated transcription of its downstream targets, we decided to further characterize this protein-protein interaction. In order to identify domains of the Pax-5 protein that are necessary for interaction with hnRNP A1, cells expressing truncated Pax-5 peptides were immunoprecipitated with an hnRNP A1 antibody, and precipitates were immunoblotted with a Pax-5 antibody. Our findings reveal that the point of contact between the two proteins is located in the Pax-5 octapeptide domain. Further, site-directed mutagenesis of Pax-5, within the octapept...
Mammaglobin 1 (MGB1) is a protein fairly specific to mammary tissues and is often over expressed ... more Mammaglobin 1 (MGB1) is a protein fairly specific to mammary tissues and is often over expressed in breast cancer. It belongs to the secretoglobin family and binds to another secretoglobin, lipophilin B (LPB), to form an active complex. The complete expression profiles of MGB1 and LPB have not been determined, however it seems certain that they are expressed in healthy mammary cells and overexpressed in cancerous mammary cells. The function of the MGB1/LPB complex is still unknown. Nonetheless, several recent studies have shed some light on one possible effect this complex could have on cancerous cells; it has been shown that the presence of MGB1 in primary breast tumors seems to reflect a less aggressive tumor phenotype and a better prognosis for this type of cancer. The objective of this study is to try to clarify the implications of the MGB1/LPB complex and its components, MGB1 and LPB individually, in breast cancer. More precisely, this study investigates the ability of the MGB1...
5-lipoxygenase (5-LO) catalyzes the initial reactions leading to the conversion of arachidonic ac... more 5-lipoxygenase (5-LO) catalyzes the initial reactions leading to the conversion of arachidonic acid into a number of biologically important mediators of inflammation. We seek to better understand the control of the biosynthesis of 5-LO products which is linked to inflammatory diseases such as asthma and atherosclerosis. In this study we used the human acute lymphocytic leukemia and Burkitt lymphoma cell lines REH and Raji, respectively, to investigate whether alternative variants of 5-LO mRNA are produced. Using reverse-transcription of extracted mRNA coupled with PCR, followed by molecular cloning and sequencing experiments, we have identified 4 different variants of 5-LO mRNA in these cell lines including the full length mRNA containing all 14 exons with the expected splicing sites. Amongst the alternative isoforms, one resulting from the exclusion of exon 13 was identified in both cell lines and was present in approximately 30% of the generated clones. This exclusion of exon 13 d...
The FASEB Journal, 2011
. 5-LO mRNA isoforms that are consistent with alternative splicing were identified by RT-PCR in a... more . 5-LO mRNA isoforms that are consistent with alternative splicing were identified by RT-PCR in a cell line or cell type-specific pattern. All evaluated cells expressed mRNA containing all 14 exons of 5-LO with the expected splicing sites. Individual isoforms that retained intron 10 (␣-10), lacked exon 13 (⌬-13), and lacked exons 10 and 13 (⌬-10,13) or that lacked the first 96 base pairs of exon 10 (⌬-p10) were identified. Immunoreactive bands coeluting with the cloned ␣-10 and ⌬-13 isoforms were measured in primary neutrophils and in Raji cells. When expressed in HEK293 cells, alternative proteins were without catalytic activity. However, when coexpressed with the active full-length 5-LO, alternative isoforms significantly decreased the biosynthesis of 5-LO products by up to 44%, as assessed by reverse-phase HPLC analysis. Additionally, in stimulated neutrophils the full-length active 5-LO was detected by immunoblot in both nuclear and nonnuclear compartments, while the ⌬-13 isoform was only detected in the nuclear fraction. These alternative 5-LO isoforms may represent a new mechanism for the regulation of the 5-LO pathway and lipid mediator biosynthesis.-Boudreau, L. H., Bertin, J., Robichaud, P. P., Laflamme, M., Ouellette, R. J., Flamand, N., Surette, M. E. Novel 5-lipoxygenase isoforms affect the biosynthesis of 5-lipoxygenase products. FASEB J. 25, 1097-1105 (2011). www.fasebj.org
OMICS: A Journal of Integrative Biology, 2007
Our understanding of gene function and gene interactions has changed dramatically with the develo... more Our understanding of gene function and gene interactions has changed dramatically with the development of high-throughput systems. It now seems clear that any given gene interacts with a number of different partners, and in a number of different molecular pathways. Traditionally, gene function has been studied using animal knockout systems or naturally occurring mutants. RNA-based gene suppression systems for example, RNA interference or ribozymes, offer a number of advantages over the traditional systems, including ease of use, high specificity, and efficacy in nearly any biological system, and the ability to perform large-scale screens. Since their advent in the mid-1990s, DNA microarrays have been the choice for genome-wide expression analysis. The synergistic effect from the combined use of RNA-based gene suppression and molecular profiling is providing researchers with vast amounts of data. As a result, we are rapidly gaining an understanding of gene interactions and function. This review will focus primarily on gene inactivation systems that have been proven worthy of use in molecular pathway analysis when combined with microarray analysis.
![Research paper thumbnail of Corrigendum to “Transcriptional response of Atlantic salmon (Salmo salar) after primary versus secondary exposure to infectious salmon anemia virus (ISAV)” [Mol. Immunol. 51 (2) (June 2012) 197–209]](https://attachments.academia-assets.com/45508853/thumbnails/1.jpg)
](Molecular Immunology, 2014
Journal of Phycology, 2009
The FASEB Journal, 2007
ABSTRACT 5-lipoxygenase (5-LO) catalyzes the initial reactions leading to the conversion of arach... more ABSTRACT 5-lipoxygenase (5-LO) catalyzes the initial reactions leading to the conversion of arachidonic acid into a number of biologically important mediators of inflammation. We seek to better understand the control of the biosynthesis of 5-LO products which is linked to inflammatory diseases such as asthma and atherosclerosis. In this study we used the human acute lymphocytic leukemia and Burkitt lymphoma cell lines REH and Raji, respectively, to investigate whether alternative variants of 5-LO mRNA are produced. Using reverse-transcription of extracted mRNA coupled with PCR, followed by molecular cloning and sequencing experiments, we have identified 4 different variants of 5-LO mRNA in these cell lines including the full length mRNA containing all 14 exons with the expected splicing sites. Amongst the alternative isoforms, one resulting from the exclusion of exon 13 was identified in both cell lines and was present in approximately 30% of the generated clones. This exclusion of exon 13 does not induce a reading frame shift but would result in a translation product missing 57 amino acids near the carboxyl terminal. Experiments are underway to determine whether the translation products of these variant isoforms are expressed and have an impact on the capacity of cells to synthesize 5-LO products. (Supported by the Canadian Institutes of Health Research and the Canada Research Chairs Program)
Journal of Fish Diseases
The infectious salmon anaemia virus (ISAV) is the causative agent of infectious salmon anaemia (I... more The infectious salmon anaemia virus (ISAV) is the causative agent of infectious salmon anaemia (ISA), an important disease of farmed Atlantic salmon (Salmo salar L.) that has resulted in a regulatory control programme for Canada and the development of international standards by the World Organisation for Animal Health (OIE).
Molecular ecology resources, Jan 21, 2016
The increasing use of high-throughput sequencing platforms has made the isolation of pure, high m... more The increasing use of high-throughput sequencing platforms has made the isolation of pure, high molecular weight DNA a primary concern for studies of a diverse range of organisms. Purification of DNA remains a significant challenge in many tissue and sample types due to various organic and inorganic molecules that coprecipitate with nucleic acids. Molluscs, for example, contain high concentrations of polysaccharides which often coprecipitate with DNA and can inhibit downstream enzymatic reactions. We modified a low-salt CTAB (MoLSC) extraction protocol to accommodate contaminant-rich animal tissues and compared this method to a standard CTAB extraction protocol and two commercially available animal tissue DNA extraction kits using oyster adductor muscle. Comparisons of purity and molecular integrity showed that our in-house protocol yielded genomic DNA generally free of contaminants and shearing, whereas the traditional CTAB method and some of the commercial kits yielded DNA unsuita...
Fish & Shellfish Immunology, 2016
The Infectious salmon anemia virus (ISAV) is an important viral disease of farmed Atlantic salmon... more The Infectious salmon anemia virus (ISAV) is an important viral disease of farmed Atlantic salmon (Salmo salar) which has caused severe financial losses for salmon farmers around the world, including Atlantic Canada. It is listed as an OIE notifiable disease and to this day eradication of infected cages remains the current practice in many countries to mitigate the spread of the virus. All strains known to display any level of virulence are characterised by deletions in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) protein compared to ISAV-HPR0 strains and are designated ISAV-HPR∆. In contrast, HPR0 strains are non-virulent and not detected by viral culture. In Atlantic Canada, improved management and husbandry practices resulted in no outbreaks caused by ISAV-HPR∆ strains from 2007 to 2012, with however ISAV-HPR0 strains (European genotype) still being detected. Starting in 2012, new ISAV-HPR∆ strains were responsible for disease outbreaks in fish farms in Nova Scotia, Newfoundland and New-Brunswick. This study was thus designed to characterize virulence associated to three of the newly identified ISAV-HPR∆ strains and investigate gene expression responses in gills of infected fish using RNA-Seq. All three ISAV-HPR∆ strains studied through the use of in vivo challenges resulted in cumulative mortalities below 40 percent and caused the differential expression of up to 1000 genes at the peak of infection compared to non-infected fish. Many genes linked to innate immunity were over-expressed; similar to what had been observed in the head-kidney through previous work. The use of RNA-Seq also enabled us to look at ISAV transcript abundance and showed that segment 8 had the highest abundance of all 8 segments of the genome. This information has given us great insight on how ISAV interacts with his host during the course of an infection.
Oncotarget, Jan 5, 2017
Pax-5, an essential transcription factor for B lymphocyte development, has been linked with the d... more Pax-5, an essential transcription factor for B lymphocyte development, has been linked with the development and progression of lymphoid cancers and carcinoma. In contrast to B-cell cancer lesions, the specific expression signatures and roles of Pax-5 in breast cancer progression are relatively unknown. In the present study, we set out to profile Pax-5 expression in mammary tissues and elucidate the cellular and molecular roles of Pax-5 in breast cancer processes. Using immunohistology on mammary tissue arrays, Pax-5 was detected in a total of 298/306 (97.6%) samples tested. Interestingly, our studies reveal that Pax-5 inhibits aggressive features and confers anti-proliferative effects in breast carcinoma cells in contrast to its oncogenic properties in B cell cancers. More precisely, Pax-5 suppressed breast cancer cell migration, invasion and tumor spheroid formation while concomitantly promoting cell adhesion properties. We also observed that Pax-5 inhibited and reversed breast can...
Journal of Phycology, 2009
Oligonucleotides, 2008
Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosp... more Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosphoramidite-method DNA synthesis are desirable for many genomics and proteomics applications. In this communication, we present a method for the deprotection of a range of N-acyl deoxyribonucleosides (T, dA Bz , dC Bz , dC Ac , dG ibu , dG PAC ) and synthetic oligodeoxyribonucleotides, ranging in length from 5-mer to 50-mer. Oligodeoxyribonucleotides were synthesized using standard amide protecting groups (dA Bz , dC Bz , dG ibu ) and phosphoramidite chemistry on cis-diol solid phase support. This deprotection method utilizes 29% aqueous ammonia solution at 170°C for 5 minutes under monomode microwave irradiation at a 20-nmole reaction scale. Reaction products were analyzed by TLC, RP-HPLC, CE, ESI-MS, real-time PCR, agarose gel electrophoresis, and by DNA uracil glycosylase (UDG) and phosphodiesterase I (PDE) enzymatic digestions.
Bioinformatics, 2004
Motivation: In the interpretation of gene expression data from a group of microarray experiments ... more Motivation: In the interpretation of gene expression data from a group of microarray experiments that include samples from either different patients or conditions, special consideration must be given to the pleiotropic and epistatic roles of genes, as observed in the variation of gene co-expression patterns.
Journal of Biological Chemistry, 2004
The transcription factor Pax-5 occupies a central role in B cell differentiation and has been imp... more The transcription factor Pax-5 occupies a central role in B cell differentiation and has been implicated in the development of B cell lymphoma. The transcriptional activation function of Pax-5 requires an intact N-terminal DNA-binding domain and is strongly influenced by the C-terminal transactivation domain. We report the identification and characterization of five human Pax-5 isoforms, which occur through the alternative splicing of exons that encode for the C-terminal transactivation domain. These isoforms arise from the inclusion or exclusion of exon 7, exon 8, and/or exon 9. Three of the Pax-5 isoforms generate novel protein sequences rich in proline, serine, and threonine amino acids that are the hallmarks of transactivation domains. The Pax-5 isoforms are expressed in peripheral blood mononuclear cells, cancerous and non-cancerous B cell lines, as well as in primary B cell lymphoma tissue. Electrophoretic mobility shift assays demonstrate that the isoforms possess specific DNA binding activity and recognize the PAX-5 consensus binding sites. In reporter assays using the CD19 promoter, the transactivation properties of the various isoforms were significantly influenced by the changes in the C-terminal protein sequence. Finally, we demonstrate, for the first time, that human Pax-5 isoform expression is modulated by specific signaling pathways in B lymphocytes.
Oligonucleotides, 2008
Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosp... more Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosphoramidite-method DNA synthesis are desirable for many genomics and proteomics applications. In this communication, we present a method for the deprotection of a range of N-acyl deoxyribonucleosides (T, dA Bz , dC Bz , dC Ac , dG ibu , dG PAC ) and synthetic oligodeoxyribonucleotides, ranging in length from 5-mer to 50-mer. Oligodeoxyribonucleotides were synthesized using standard amide protecting groups (dA Bz , dC Bz , dG ibu ) and phosphoramidite chemistry on cis-diol solid phase support. This deprotection method utilizes 29% aqueous ammonia solution at 170°C for 5 minutes under monomode microwave irradiation at a 20-nmole reaction scale. Reaction products were analyzed by TLC, RP-HPLC, CE, ESI-MS, real-time PCR, agarose gel electrophoresis, and by DNA uracil glycosylase (UDG) and phosphodiesterase I (PDE) enzymatic digestions.
Pax-5 is an important contributor to B cell development. Several studies have shown that the dere... more Pax-5 is an important contributor to B cell development. Several studies have shown that the deregulated expression of Pax-5 is implicated in onset of different types of cancers. Previously, we have observed that Pax-5 expression in B-cells increases dramatically upon estrogen stimulation, and that Pax-5 is expressed in breast cancer cell lines while it is undetectable in normal mammary cell lines. Based on this evidence, we hypothesis that Pax-5 may exercise a key role in mammary oncogenesis and that this abnormal function may be modulated by estrogen. In order to shed light on these initial findings, using quantitative real-time RT-PCR we have characterized the Pax-5 expression profiles in several mammary cell lines following stimulation with estrogen and Tamoxifen. We have found that in each case, Pax-5 expression is differentially modulated when comparing treated and untreated cells. Further to this, we are using oligonucleotide microarrays to identify the gene expression pathwa...
The transcription factor Pax5 possesses oncogenic properties and is often deregulated in many sub... more The transcription factor Pax5 possesses oncogenic properties and is often deregulated in many subsets of B-cell lymphomas and leukemias. Furthermore, the Pax5 transcript is known to be alternatively spliced, producing a wide variety of isoforms. It has been recently suggested that deregulation of Pax5 isoforms may lead to these B-cell malignancies. Since aberrant splicing is linked to human diseases, we hypothesized that the deregulation of Pax5 isoforms may result from a defective splicing mechanism caused by mutations in splice sites. Therefore, we have attempted to establish a link between the pattern of expressed Pax5 isoforms in B-cell chronic lymphocytic leukemia (B-CLL) patients and mutations in splice sites. These experiments were performed using PCR amplification of Pax5 in B-CLL patients in conjunction with immunodetection of the protein isoforms by Western blot. Sequencing of PCR products is then used to identify mutations. Our results indicate that full length Pax5 is pr...
Pax-5 is a key regulator of B lymphocyte development, and is known to interact directly or indire... more Pax-5 is a key regulator of B lymphocyte development, and is known to interact directly or indirectly with a large number of protein partners. In order to identify proteins that interact directly with Pax-5, a yeast-two hybrid assay was performed using Pax-5 as bait. A novel interaction between Pax-5 and human nuclear ribonucleoprotein A1 (hnRNP A1) was identified. As hnRNP A1 is a protein involved in RNA processing and could influence Pax-5- mediated transcription of its downstream targets, we decided to further characterize this protein-protein interaction. In order to identify domains of the Pax-5 protein that are necessary for interaction with hnRNP A1, cells expressing truncated Pax-5 peptides were immunoprecipitated with an hnRNP A1 antibody, and precipitates were immunoblotted with a Pax-5 antibody. Our findings reveal that the point of contact between the two proteins is located in the Pax-5 octapeptide domain. Further, site-directed mutagenesis of Pax-5, within the octapept...
Mammaglobin 1 (MGB1) is a protein fairly specific to mammary tissues and is often over expressed ... more Mammaglobin 1 (MGB1) is a protein fairly specific to mammary tissues and is often over expressed in breast cancer. It belongs to the secretoglobin family and binds to another secretoglobin, lipophilin B (LPB), to form an active complex. The complete expression profiles of MGB1 and LPB have not been determined, however it seems certain that they are expressed in healthy mammary cells and overexpressed in cancerous mammary cells. The function of the MGB1/LPB complex is still unknown. Nonetheless, several recent studies have shed some light on one possible effect this complex could have on cancerous cells; it has been shown that the presence of MGB1 in primary breast tumors seems to reflect a less aggressive tumor phenotype and a better prognosis for this type of cancer. The objective of this study is to try to clarify the implications of the MGB1/LPB complex and its components, MGB1 and LPB individually, in breast cancer. More precisely, this study investigates the ability of the MGB1...
5-lipoxygenase (5-LO) catalyzes the initial reactions leading to the conversion of arachidonic ac... more 5-lipoxygenase (5-LO) catalyzes the initial reactions leading to the conversion of arachidonic acid into a number of biologically important mediators of inflammation. We seek to better understand the control of the biosynthesis of 5-LO products which is linked to inflammatory diseases such as asthma and atherosclerosis. In this study we used the human acute lymphocytic leukemia and Burkitt lymphoma cell lines REH and Raji, respectively, to investigate whether alternative variants of 5-LO mRNA are produced. Using reverse-transcription of extracted mRNA coupled with PCR, followed by molecular cloning and sequencing experiments, we have identified 4 different variants of 5-LO mRNA in these cell lines including the full length mRNA containing all 14 exons with the expected splicing sites. Amongst the alternative isoforms, one resulting from the exclusion of exon 13 was identified in both cell lines and was present in approximately 30% of the generated clones. This exclusion of exon 13 d...
The FASEB Journal, 2011
. 5-LO mRNA isoforms that are consistent with alternative splicing were identified by RT-PCR in a... more . 5-LO mRNA isoforms that are consistent with alternative splicing were identified by RT-PCR in a cell line or cell type-specific pattern. All evaluated cells expressed mRNA containing all 14 exons of 5-LO with the expected splicing sites. Individual isoforms that retained intron 10 (␣-10), lacked exon 13 (⌬-13), and lacked exons 10 and 13 (⌬-10,13) or that lacked the first 96 base pairs of exon 10 (⌬-p10) were identified. Immunoreactive bands coeluting with the cloned ␣-10 and ⌬-13 isoforms were measured in primary neutrophils and in Raji cells. When expressed in HEK293 cells, alternative proteins were without catalytic activity. However, when coexpressed with the active full-length 5-LO, alternative isoforms significantly decreased the biosynthesis of 5-LO products by up to 44%, as assessed by reverse-phase HPLC analysis. Additionally, in stimulated neutrophils the full-length active 5-LO was detected by immunoblot in both nuclear and nonnuclear compartments, while the ⌬-13 isoform was only detected in the nuclear fraction. These alternative 5-LO isoforms may represent a new mechanism for the regulation of the 5-LO pathway and lipid mediator biosynthesis.-Boudreau, L. H., Bertin, J., Robichaud, P. P., Laflamme, M., Ouellette, R. J., Flamand, N., Surette, M. E. Novel 5-lipoxygenase isoforms affect the biosynthesis of 5-lipoxygenase products. FASEB J. 25, 1097-1105 (2011). www.fasebj.org
OMICS: A Journal of Integrative Biology, 2007
Our understanding of gene function and gene interactions has changed dramatically with the develo... more Our understanding of gene function and gene interactions has changed dramatically with the development of high-throughput systems. It now seems clear that any given gene interacts with a number of different partners, and in a number of different molecular pathways. Traditionally, gene function has been studied using animal knockout systems or naturally occurring mutants. RNA-based gene suppression systems for example, RNA interference or ribozymes, offer a number of advantages over the traditional systems, including ease of use, high specificity, and efficacy in nearly any biological system, and the ability to perform large-scale screens. Since their advent in the mid-1990s, DNA microarrays have been the choice for genome-wide expression analysis. The synergistic effect from the combined use of RNA-based gene suppression and molecular profiling is providing researchers with vast amounts of data. As a result, we are rapidly gaining an understanding of gene interactions and function. This review will focus primarily on gene inactivation systems that have been proven worthy of use in molecular pathway analysis when combined with microarray analysis.
Molecular Immunology, 2014
Journal of Phycology, 2009