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Characterization of the genes encoding tropomyosin and ARP4 in Neurospora crassa /Haghighi, Nahideh, January 1900 (has links)
Thesis (M. Sc.)--Carleton University, 2004. / Includes bibliographical references (p. 118-131). Also available in electronic format on the Internet.
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Su(un-24)-1, a suppressor of a temperature sensitive ribonucleotide reductase mutation in neurospora crassa : characterization and PCR-based mapping /Kotierk, Moshi January 1900 (has links)
Thesis (M. Sc.)--Carleton University, 2004. / Includes bibliographical references (p. 79-85). Also available in electronic format on the Internet.
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Gel-mobility assays of cysteine mutants in the C-terminus region of the Neurospora crassa large subunit of ribonucleotide reductase /Siahbazi, Mojgan, January 1900 (has links)
Thesis (M.Sc.) - Carleton University, 2008. / Includes bibliographical references (p. 70-75). Also available in electronic format on the Internet.
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Molecular genetic studies of oligodendroglial and ependymal tumors.January 1998 (has links)
by Tong Yuen Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 124-141). / Abstract also in Chinese. / acknowledgements --- p.i / Abstract (English/Chinese) --- p.ii / contents --- p.vi / list of tables --- p.viii / ost of figures --- p.x / Chapter I. --- introduction --- p.1 / Chapter I.1. --- Tumors of the Central Nervous System --- p.1 / Chapter I.2. --- Histopathological Classification of Human Glial Tumors --- p.3 / Chapter I.2.1. --- Histopathology of Astrocytic Gliomas --- p.3 / Chapter I.2.1.1. --- Diffuse Astrocytomas --- p.3 / Chapter I.2.1.2. --- Others --- p.6 / Chapter I.2.2. --- Histopathology of Non-Astrocytic Gliomas --- p.6 / Chapter I.2.2.1. --- Oligodendroglial Tumors --- p.6 / Chapter I.2.2.2. --- Ependymal Tumors --- p.9 / Chapter I.3. --- Tumor Suppressor Genes --- p.14 / Chapter I.3.1. --- p53 --- p.14 / Chapter I.3.1.1. --- Historical Perspectives --- p.14 / Chapter I.3.1.2. --- Structure of p53 Gene and Protein --- p.15 / Chapter I.3.1.3. --- Functions of Wild-Type p53 Protein --- p.18 / Chapter I.3.1.4. --- Regulation and Modulation of the Functions of p53 --- p.21 / Chapter I.3.1.5. --- Mechnism of p53 Inactivation --- p.23 / Chapter I.3.1.6. --- p53 Mutation Profiles in Human Tumors --- p.25 / Chapter I.3.2. --- Novel Genes --- p.28 / Chapter I.3.2.1. --- PTEN/MMAC1 --- p.28 / Chapter I.3.2.2. --- DMBT1 --- p.31 / Chapter I.4. --- Cytogenetic and Molecular Genetic Studies in Gliomas --- p.34 / Chapter I.4.1. --- Astrocytic Gliomas --- p.34 / Chapter I.4.2. --- Non-Astrocytic Gliomas --- p.39 / Chapter I.4.2.1. --- Oligodendroglial Tumors --- p.39 / Chapter I.4.2.2. --- Ependymal Tumors --- p.46 / Chapter II. --- objectives of study --- p.49 / Chapter III. --- materials and methods --- p.52 / Chapter III.l. --- Patients and Materials --- p.52 / Chapter III.2. --- Collection of Samples --- p.57 / Chapter III.3. --- DNA Extraction --- p.58 / Chapter III.3.1. --- Extraction of Genomic DNA from Formalin-Fixed Paraffin Embedded Tissues --- p.58 / Chapter III.3.2. --- Extraction of Genomic DNA from Blood --- p.60 / Chapter III.4. --- Loss of Heterozygosity (LOH) Analysis on Chromosome 10q --- p.61 / Chapter III.4.1. --- Microsatellite Markers --- p.62 / Chapter III.4.2. --- Amplification of Target Sequences by PCR --- p.63 / Chapter III.4.3. --- Denaturing Polyaerylamide Gel Electrophoresis --- p.64 / Chapter III.4.4. --- Detection of Loss of Heterozygosity (LOH) --- p.64 / Chapter III.5. --- Mutational Analysis of p53 and PTEN/MMAC1 --- p.66 / Chapter III.5.1. --- Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) Analysis --- p.66 / Chapter III.5.1.1. --- PCR Primers --- p.66 / Chapter III.5.1.2. --- PCR Amplification of Target Sequences --- p.68 / Chapter III.5.1.3. --- Non-denaturing Polyacrylamide Gel Electrophoresis --- p.71 / Chapter III.5.2. --- Direct DNA Sequencing Analysis --- p.72 / Chapter III.5.2.1. --- Cycle Sequencing --- p.72 / Chapter III.5.2.2. --- Denaturing Gel Electrophoresis --- p.73 / Chapter III.6. --- Differential PCR for Detection of MDM2 Amplification --- p.74 / Chapter III.6.1. --- DNA Amplification by PCR --- p.74 / Chapter III.6.2. --- Polyacrylamide Gel Electrophoresis --- p.75 / Chapter III.6.3. --- Detection of Gene Amplification --- p.75 / Chapter IV. --- Results --- p.77 / Chapter IV.1. --- LOH Analysis of Chromosome l0q --- p.77 / Chapter IV.2. --- Mutational Analysis ofp53 and PTEN/MMAC1 --- p.92 / Chapter IV.3. --- Differential PCR Analysis of MDM2 Amplification --- p.103 / Chapter V. --- discussion --- p.109 / Chapter V.l. --- p53 Gene Inactivation Studies --- p.110 / Chapter V.2. --- Molecular Genetic Studies on Chromosome l0q --- p.113 / Chapter V.3. --- Microsatellite Instability in Non-Astrocytic Gliomas --- p.117 / Chapter V.4. --- Significance of This Study --- p.118 / Chapter V.5. --- Limitations of This Study --- p.119 / Chapter V.6. --- Future Studies --- p.122 / Chapter VI. --- REFERENCES --- p.124
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Mutation induction characteristics and parameters of antibiotic stress.January 2010 (has links)
Wong, Ah Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (p. 125-130). / Abstracts in English and Chinese. / ABSTRACT --- p.V / 摘要 --- p.VIII / TABLE OF CONTENTS --- p.X / ACKNOWLEDGEMENTS --- p.XII / INTRODUCTION --- p.13 / Adaptive Mutation versus Spontaneous Mutation --- p.13 / Fluctuation Test --- p.13 / Adaptive Reversion of lacI - lacZ Fusion Mutant --- p.17 / Putative Models of Adaptive Mutation Mechanisms --- p.18 / Point Mutagenesis --- p.18 / Hypermutation --- p.19 / Gene Amplification --- p.21 / Controversy over the Mechanism of Adaptive Mutation --- p.21 / Induced Mutagenesis under Other Stresses --- p.23 / The General Stress Response --- p.23 / The SOS Response --- p.24 / Reduced Mismatch Repair --- p.24 / Adaptive Mutation in Other Micro-organisms --- p.25 / Mutation Generation under Antibiotic Stress --- p.26 / Fluoroquinolones --- p.26 / Beta-Lactams --- p.27 / Aminoglycosides --- p.27 / Justification and Objectives of this Study --- p.28 / MATERIAL AND METHODS --- p.30 / Bacterial Strains --- p.30 / Culture Media --- p.30 / Antibiotics --- p.30 / Resistance Induction Assay --- p.31 / Rationale of Experimental Design --- p.31 / Agar Selection Method --- p.32 / Broth Selection Method --- p.34 / Isolation of Organisms which Exhibited Reduced Drug Susceptibility and Determination the Minimal Inhibitory Concentration (MIC) --- p.36 / Indole Test --- p.37 / DNA Extraction --- p.37 / Polymerase Chain Reaction (PCR) on the gyrA and rpoB Genes --- p.37 / PCR Product Purification and Nucleotide Sequencing --- p.39 / RESULTS --- p.40 / Solid Agar Selection Approach --- p.41 / Broth Selection Approach --- p.48 / Strain MG1655 --- p.49 / Strain BW25113 --- p.53 / recA Deletion Mutant --- p.54 / mutS Deletion Mutant --- p.56 / DISCUSSION --- p.59 / Development of Resistance Induction Assay --- p.59 / Background Resistance to Gentamicin and Rifampicin --- p.62 / Resistance Induction Effect of Ciprofloxacin --- p.64 / Relative Effects of recA and mutS Deletion --- p.66 / Putative Origins of Antibiotic Resistance Gene Mutations --- p.69 / TABLES AND FIGURES --- p.71 / REFERENCES --- p.125
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Evolution Of New Metabolic Functions By Mutations In Pre-Existing Genes : The chb Operon Of Escherichia Coli As A ParadigmKachroo, Aashiq Hussain 02 1900 (has links)
Escherichia coli has the ability to respond to stress such as starvation in a very efficient manner. Under conditions of starvation wherein a novel substrate is provided as a sole nutritional source,
Spontaneous mutants arise in a population of E.Coli that are able to utilize this novel carbon Many generic systems, upon mutational activation, have been shown to allow E.coli to Grow on novel substrates. .
Wildtype E.coli is not able to utilize cellobiose, a disaccharide of glucose, as a carbon source. However after prolonged incubation with cellobiose as a sole carbon source, spontaneous Cel+ mutants can be isolated. The Cel+ derivatives have mutations in the chb operon involved in the utilization of N-N-diacetylchitobiose, a disaccharide of N-acetyl glucosamine. The chb operon of E.coli is comprised of six ORFs (chbBCARFG) with a ~200bp regulatory region (chbOP); chbBCA encode the IIB, IIC and IIA domains of the PTS-dependent permease respectively, chbR encodes for a dual function activator/repressor, chbF
encodes the phopho-chitobiase and chbG codes for a protein of unknown function. It has been shown that the three proteins ChbR, CAP and NagC regulate the expression of the chb operon. ChbR along with CAP activates the chb operon in the presence of chitobiose. In the absence of the inducer, ChbR, along with NagC, represses the chb operon.
Activation of the chb operon allowing utilization of cellobiose was earlier shown to occur either via insertion of IS1, IS2 or IS5 into the regulatory region (chbOP) upstream of the transcription start site or by base substitutions in chbR. Comparison of the chb operon sequence obtained from various Cel+ mutants with E.coli K12 genome sequence showed many differences. These differences were clustered in both the permease (chbBCA) as well as the enzyme (chbF) of the chb operon, suggesting that mutations are needed in all the ORFs of this
operon in order to alter the specificity of E.coli towards utilization of cellobiose. The main objective of this thesis is to elucidate the mechanism of mutational activation of the chb operon of E.coli to allow utilization of cellobiose. These studies have shown that two classes of
mutations, those that abrogate repression by NagC and those that alter the regulation by
ChbR, together are necessary and sufficient to confer a Cel+ phenotype to E.coli. These studies also show that the wildtype permease and phospho-â -glucosidase are able to recognize and cleave cellobiose.
Initial experiments were designed to study the role of independent mutational events
of either insertion within the regulatory region or loss-of-function of chbR in conferring E.coli a Cel+ phenotype. The single mutational event of either the insertion within the regulatory region chbOP that disrupts the strong NagC binding site (mimicking an IS element) or knockout of chbR did not confer on E.c oli a Cel+ phenotype. However the presence of the
artificial insertion within chbOP accelerated the process of obtaining Cel+ mutants suggesting a positive role for insertion elements. The apparent inability of the chbR knockout strain to mutate to Cel+ suggested that chbR is essential for acquisition of a Cel+ phenotype. Reporter
gene assays showed that the presence of an insertion within chbOP enhances the promoter
activity marginally. The role of chbR as a repressor was further ascertained by increased promoter activity seen from wildtype chbOP-lacZ fusion in a chbR knockout strain. A marginal enhancement in promoter activity in the presence of cellobiose in a strain carrying a wildtype
chbR as compared to chbR knockout strain suggested an additional positive role of chbR. The inability of cellobiose to produce an inducing signal necessary for activation by wildtype ChbR protein suggested that gain-of-function mutations within chbR locus might play a crucial role in acquisition of cellobiose utilization phenotype by E.coli.
The chbR clones obtained from various Cel+ mutants could activate transcription from
the chb promoter at a higher level in the presence of cellobiose. However this activation was seen only in a strain carrying disruptions of the chromosomal nagC and chbR loci. These transformants also showed a Cel+ phenotype on the MacConkey cellobiose medium suggesting that the wildtype permease and enzyme upon induction could recognise, transport and cleave
cellobiose, respectively. This was confirmed by cloning the wildtype genes encoding the
permease and phospho-â -glucosidase under a heterologous promoter (Plac). The wildtype
E.coli strain transformed with a plasmid carrying the genes could utilize cellobiose efficiently.
Large scale isolation of Cel+ mutants was undertaken. Variation in the ability of
cellobiose utilization was observed among the different mutants. Several Cel+ mutants retained the ability to utilize chitobiose. Cel+ mutants lacking insertions within chbOP contained a loss-of-function mutation at the nagC locus. The sequencing of the chbR locus from Cel+ mutant strains showed a single basepair change at the DNA level translating into a single amino acid change when compared to the Cel- counterpart. Nucleotide sequence of chbR obtained from
two Cel+ natural isolates of E.coli also showed a single base mutation. The chbR clones from the two mutants, when transformed into a strain carrying disruptions at the chromosomal nagC
and chbR loci, conferred it a Cel+ phenotype.
Initial characterization of one of the mutant ChbR (N238S) was carried out. Reporter assays in a strain containing a wildtype copy of chbR at the genomic locus and a disruption of nagC showed that the wildtype ChbR is dominant over the mutant ChbR (N238S). The biochemical investigations of the wildtype and mutant ChbR (N238S) were undertaken. Wildtype ChbR showed non-specific binding to chbOP that could not be competed out by excess cold DNA. DNaseI protection assays confirmed that wildtype ChbR formed a relatively nonspecific complex with chbOP as compared to mutant ChbR (N238S). Finally DNaseI footprinting experiments showed that mutant ChbR (N238S) binds the specific direct repeat within chbOP better than the wildtype protein. These results indicated that mutant ChbR
(N238S) has lost its ability to repress transcription by its inability to bind chbOP non-specifically. In addition, the mutant ChbR (N238S) has acquired the ability to activate transcription in the presence of cellobiose. This could be partly mediated via enhanced binding of the mutant ChbR (N238S) to the specific DNA binding site within chbOP in contrast to its wildtype counterpart.
To conclude, this work has shown that acquisitive evolution of E.coli towards
utilization of cellobiose in laboratory conditions alters the regulation of the chb operon and allows it to acquire new metabolic capability for utilizing cellobiose under selective pressure.
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Estudo de mutações no gene BRCA na população Ashkenazi e não Ashkenazi com histórico para câncer de mama e/ou ovário. / Study of mutations in the BRCA genes in Ashkenazi and non-Ashkenazi jewish population with familial breast and/or ovarian cancer.Cunha, Danielle Renzoni da 07 April 2011 (has links)
O câncer de mama é um dos mais incidentes no mundo e mais comum na população feminina. Algumas populações possuem maior risco para o câncer de mama e ovário devido a presença de mutações fundadoras nos genes BRCA1 e BRCA2 como ocorre nos Judeus Ashkenazi (JA). Os testes genéticos para detecção de mutações de ponto nos genes BRCA1 e BRCA2 foram realizados em 106 indivíduos com e sem origem judaica (IOA) através do rastreamento por dHPLC e sequenciamento. A distribuição das mutações fundadoras no gene BRCA1 foi de cerca de 55 % para 185delAG e 5382isnC no gene BRCA1 e 12 % para 6174delT, no gene BRCA2. Estas mutações também foram detectadas em 7,4 % dos casos no grupo IOA e 84,2% no grupo JA (p< 0,001). Mutações fundadoras espanholas e portuguesas, 330 A>G (11,11 %) e 7966C>T (7,4 %), foram detectadas no IOA. Os carcinomas de mama e ovário foram mais frequentes nos dois grupos, cerca de 70 % e 20 %, respectivamente. No grupo IOA foram diagnosticados carcinomas de mama masculino (9,5 %) e trompa uterina (2,9 %), e carcinoma endometrióide em 11,8 % no grupo JA. / Breast cancer is one of the most commun tumors in women. Some populations shows higher risks for breast and ovarian cancers due to founder mutations in the BRCA1 and BRCA2 genes, as well as Ashkenazi Jewish (AJ) population. Genetic tests to detect point mutations in the BRCA1 and BRCA2 genes were made in 106 individuals: Ashkenazi Jewish and non-Ashkenazi Jews (NAJ) using dHPLC screening and sequencing. The distribution of founder mutations in the BRCA1 and BRCA2. genes was about 55 % for 185delAG and 5382isnC in BRCA1 gene and 12 % for 6174delT in BRCA2 gene. Those mutations were also detected in 7,4 % of NAJ group and 84,2 % in AJ group (p<0,001). Spanish and portuguese founder mutations 330 A>G (11,11 %) and 7966C>T (7,4 %) were also detected in NAJ group. Breast and ovarian carcinomas were detected in higher frequencies in both groups, about 70 % and 20 %, respectively. Male breast carcinomas (9,5 %) and uterine tube carcinomas ( 2,9 %) were diagnosed in NAJ group, and endometrioid carcinoma in AJ group (11, 8 %).
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Estudo de mutações no gene BRCA na população Ashkenazi e não Ashkenazi com histórico para câncer de mama e/ou ovário. / Study of mutations in the BRCA genes in Ashkenazi and non-Ashkenazi jewish population with familial breast and/or ovarian cancer.Danielle Renzoni da Cunha 07 April 2011 (has links)
O câncer de mama é um dos mais incidentes no mundo e mais comum na população feminina. Algumas populações possuem maior risco para o câncer de mama e ovário devido a presença de mutações fundadoras nos genes BRCA1 e BRCA2 como ocorre nos Judeus Ashkenazi (JA). Os testes genéticos para detecção de mutações de ponto nos genes BRCA1 e BRCA2 foram realizados em 106 indivíduos com e sem origem judaica (IOA) através do rastreamento por dHPLC e sequenciamento. A distribuição das mutações fundadoras no gene BRCA1 foi de cerca de 55 % para 185delAG e 5382isnC no gene BRCA1 e 12 % para 6174delT, no gene BRCA2. Estas mutações também foram detectadas em 7,4 % dos casos no grupo IOA e 84,2% no grupo JA (p< 0,001). Mutações fundadoras espanholas e portuguesas, 330 A>G (11,11 %) e 7966C>T (7,4 %), foram detectadas no IOA. Os carcinomas de mama e ovário foram mais frequentes nos dois grupos, cerca de 70 % e 20 %, respectivamente. No grupo IOA foram diagnosticados carcinomas de mama masculino (9,5 %) e trompa uterina (2,9 %), e carcinoma endometrióide em 11,8 % no grupo JA. / Breast cancer is one of the most commun tumors in women. Some populations shows higher risks for breast and ovarian cancers due to founder mutations in the BRCA1 and BRCA2 genes, as well as Ashkenazi Jewish (AJ) population. Genetic tests to detect point mutations in the BRCA1 and BRCA2 genes were made in 106 individuals: Ashkenazi Jewish and non-Ashkenazi Jews (NAJ) using dHPLC screening and sequencing. The distribution of founder mutations in the BRCA1 and BRCA2. genes was about 55 % for 185delAG and 5382isnC in BRCA1 gene and 12 % for 6174delT in BRCA2 gene. Those mutations were also detected in 7,4 % of NAJ group and 84,2 % in AJ group (p<0,001). Spanish and portuguese founder mutations 330 A>G (11,11 %) and 7966C>T (7,4 %) were also detected in NAJ group. Breast and ovarian carcinomas were detected in higher frequencies in both groups, about 70 % and 20 %, respectively. Male breast carcinomas (9,5 %) and uterine tube carcinomas ( 2,9 %) were diagnosed in NAJ group, and endometrioid carcinoma in AJ group (11, 8 %).
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Relative contributions of the stringent response mediators (p)ppGpp and DksA to Haemophilus ducreyi virulence in humansHolley, Concerta Leigh 17 June 2015 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcerative disease that facilitates the transmission of HIV-1. H. ducreyi also causes non-sexually transmitted cutaneous ulcers in children in tropical regions. During human infection, H. ducreyi is subject to a variety of stresses. The stringent response is a bacterial stress response system induced by nutrient limiting conditions and mediated by guanosine tetra- and pentaphosphate [(p)ppGpp] and the transcriptional regulator DksA. (p)ppGpp and DksA jointly interact with RNA polymerase to regulate genes critical for bacterial survival. We hypothesized that the stringent response is required for H. ducreyi virulence in humans. A ΔrelAΔspoT mutant, which is unable to synthesize (p)ppGpp, was partially attenuated for abscess formation in human volunteers. Loss of (p)ppGpp increased bacterial resistance to phagocytosis and stationary phase survival; however, the mutant was more sensitive to oxidative stress. A ΔdksA mutant was also partially attenuated in humans. The ΔdksA mutant behaved like the (p)ppGpp mutant in stationary phase survival and sensitivity to oxidative stress, but exhibited decreased resistance to phagocytosis. Both mutants had decreased adherence to fibroblasts, but the mechanisms underlying the adherence defect were distinct. To better understand the roles of (p)ppGpp and DksA in regulating gene expression, we performed transcriptome analysis of the parent and mutant strains. (p)ppGpp and DksA deficiency resulted in dysregulation of multiple genes including several known virulence determinants. At stationary phase, (p)ppGpp and DksA targets were not identical but significantly overlapped; as the mutants were phenotypically distinct, this finding underscores both the unique and joint roles DksA and (p)ppGpp play in regulation of H. ducreyi virulence. We conclude that (p)ppGpp and DksA play significant roles in H. ducreyi pathogenesis. This is the first study to show that the stringent response has a direct role in the ability of a bacterial pathogen to cause disease in humans.
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Estudos dos genes Tbx19 e Crhr1 em cães da raça poodle com hipercortisolismo ACTH-dependente / Study of Tbx19 and Crhr1 genes in Poodle dogs with ACTH-dependent hypercortisolismMarco, Viviani de 29 April 2010 (has links)
O hipercortisolismo ACTH-dependente (HAD), também chamado de doença de Cushing, é uma das endocrinopatias mais comumente diagnosticadas na espécie canina. A sintomatologia clínica ocorre, secundariamente, aos efeitos gliconeogênicos, catabólicos, antiinflamatórios e imunossupressores dos glicocorticóides sobre vários sistemas orgânicos. Há uma marcante predisposição da doença na raça poodle e casos familiais têm sido diagnosticados sugerindo uma causa genética. As alterações moleculares que levam ao desenvolvimento do HAD em cães permanecem indefinidas. Dentre os genes implicados no desenvolvimento dos corticotrofos e na regulação do eixo corticotrófico, destacam-se o Tbx19 e o Crhr1, respectivamente. O Tbx19 é um fator de transcrição obrigatório para a transcrição do gene da proopiomelanocortina (POMC) e para a diferenciação terminal dos corticotrofos. Como está presente, exclusivamente, em corticotrofos normais e adenomatosos, foi proposto seu envolvimento na secreção excessiva de ACTH na doença de Cushing. A presença de CRHR1 nos corticotrofinomas na espécie humana e canina levantou a hipótese da sua participação na tumorigênese hipofisária, promovendo uma estimulação celular prolongada, mesmo na ausência de hormônios hipotalâmicos. Um aumento da expressão do CRHR1 foi demonstrado nos tumores corticotróficos, apesar da secreção autônoma de ACTH e dos níveis portais suprimidos de CRH em pacientes humanos e caninos com doença de Cushing. Os objetivos do presente trabalho foram pesquisar a presença de mutações germinativas nas regiões codificadoras dos genes Tbx19 e Crhr1 em cães com HAD. Para tanto, estudamos 50 cães da raça poodle com hipercortisolismo ACTH-dependente (33 fêmeas e 17 machos), com idade média de 8,71 anos e 50 cães controle da mesma raça (32 fêmeas e 18 machos) com idade superior a 6 anos (média de 9,38 anos) e sem endocrinopatias. O DNA genômico foi extraído e amplificado através da reação de polimerização em cadeia (PCR), utilizando-se oligonucleotídeos (primers) específicos para os genes Tbx19 e Crhr1. Foi identificada uma nova variante alélica tanto no Tbx19 como no Crhr1, ambas não descritas na literatura. No gene Tbx19, a variante p. S343G foi encontrada em dois cães não aparentados, mas também em dois controles normais, sugerindo tratar-se de um novo polimorfismo. Já a variante p. V97M do Crhr1 foi encontrada, em heterozigose em um animal com HAD, porém não foi observada em cem alelos normais. O códon 97 está localizado no domínio extracelular aminoterminal do gene Crhr1, de extrema importância para a ligação com alta afinidade ao ligante. O estudo molecular da estrutura quartenária da proteína mutada, seguido da avaliação da energia de ligação da superfície de contato entre o hormônio e o receptor revelou um rearranjo estrutural com alteração da superfície de contato entre o CRH e o seu receptor CRHR1, resultando em uma energia de ligação 17% superior à do receptor selvagem. Em conclusão, esse estudo não identificou alterações no gene Tbx19 associadas ao hipercortisolismo ACTH-dependente canino, mas por outro lado, identificou pela primeira vez, uma mutação ativadora no Crhr1, provavelmente responsável pelo hipercortisolismo ACTH-dependente em um cão da raça poodle. / The ACTH-dependent hypercortisolism (ADH), also called Cushing\'s disease, is one of the most commonly diagnosed endocrine diseases in dogs. The symptoms occur due to glucocorticoids excess leading to gluconeogenic, catabolic, anti-inflammatory and immunosuppressive effects in multiple organs and systems. There is a high incidence of Cushing\'s disease in Poodles and familial disease has been identified suggesting a genetic involvement. The molecular changes that lead to the development of ACTH-dependent hypercortisolism in dogs remain undefined. Among genes implicated in corticotroph development and in corticotropic axis regulation, we would like to point out Tbx19 and Crhr1, respectively. Tbx19 gene is a transcription factor required for transcription of the proopiomelanocortin gene and for terminal differentiation of the corticotroph. Inactivating mutations in that gene are associated with human isolated ACTH deficiency. Since Tbx19 is present exclusively in normal and adenomatous corticotroph cells, its involvement in the secretion of ACTH in Cushing\'s disease was proposed. The presence of CRHR1 in corticotrophinomas in humans and dogs raised the possibility of its involvement in pituitary tumorigenesis, promoting prolonged cell stimulation, even in the absence of hypothalamic hormones. An increased expression of the CRHR1 mRNA was demonstrated in human and canine ACTH-secreting pituitary adenomas, despite the autonomous ACTH secretion and the low portal levels of CRH. The aim of this study was to investigate Tbx19 and Crhr1 coding region mutations in Poodle dogs with ACTH-dependent hypercortisolism. We studied 50 Poodle dogs with ADH (33 females and 17 males) with a mean age of 8.71 years and 50 control dogs of the same breed (32 females and 18 males) older than 6 years (mean 9.38 years) and without endocrinopathies. Genomic DNA was extracted from peripheral blood, amplified by the polymerase chain reaction (PCR) using specific intronic primers and submitted to automatic sequence. We identified a new allelic variant in the Tbx19 and Crhr1 coding regions. The allelic variant p. S343G in the Tbx19 gene was found in two unrelated dogs, but also in two normal controls, suggesting that this is a new polymorphism. The Crhr1 allelic variant p. V97M was found in heterozygosity in one animal with ACTH-dependent hypercortisolism, but was not observed in one hundred normal alleles. The codon 97 is located in the extracellular amino terminal domain of the Crhr1 and is extremely important for high affinity ligand binding. The molecular analysis of the quaternary structure of normal and mutated proteins, followed by evaluation of the binding energy of the contact surface between the hormone and the receptor showed a structural rearrangement of the mutated protein by changing the contact surface between the CRH and its receptor CRHR1, resulting in a binding energy 17% higher than the wild type. In conclusion, this study did not identify Tbx19 mutations associated with canine ACTH-dependent hypercortisolism, but on the other hand, we first identified a Crhr1 gain-of-function mutation probably responsible for ACTH-dependent hypercortisolism in a Poodle dog of our cohort.
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