RESUMO
MRN-MDC1 plays a central role in the DNA damage response (DDR) and repair. Using proteomics of isolated chromatin fragments, we identified DDR factors, such as MDC1, among those highly associating with a genomic locus upon transcriptional activation. Purification of MDC1 in the absence of exogenous DNA damage revealed interactions with factors involved in gene expression and RNA processing, in addition to DDR factors. ChIP-seq showed that MRN subunits, MRE11 and NBS1, colocalized throughout the genome, notably at TSSs and bodies of actively transcribing genes, which was dependent on the RNAPII transcriptional complex rather than transcription per se. Depletion of MRN increased RNAPII abundance at MRE11/NBS1-bound genes. Prolonged MRE11 or NBS1 depletion induced single-nucleotide polymorphisms across actively transcribing MRN target genes. These data suggest that association of MRN with the transcriptional machinery constitutively scans active genes for transcription-induced DNA damage to preserve the integrity of the coding genome.
Assuntos
Proteínas de Ciclo Celular , Cromatina , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Humanos , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
Expression from the HIV-1 LTR can be repressed in a small population of cells, which contributes to the latent reservoir. The factors mediating this repression have not been clearly elucidated. We have identified a network of nuclear RNA surveillance factors that act as effectors of HIV-1 silencing. RRP6, MTR4, ZCCHC8 and ZFC3H1 physically associate with the HIV-1 TAR region and repress transcriptional output and recruitment of RNAPII to the LTR. Knock-down of these factors in J-Lat cells increased the number of GFP-positive cells, with a concomitant increase in histone marks associated with transcriptional activation. Loss of these factors increased HIV-1 expression from infected PBMCs and led to reactivation of HIV-1 from latently infected PBMCs. These findings identify a network of novel transcriptional repressors that control HIV-1 expression and which could open new avenues for therapeutic intervention.
Assuntos
Infecções por HIV/virologia , Repetição Terminal Longa de HIV/genética , HIV-1/genética , Proteínas Nucleares/metabolismo , RNA Nuclear/metabolismo , Proteínas Repressoras/metabolismo , Ativação Viral , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Regulação Viral da Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/metabolismo , HIV-1/patogenicidade , Células HeLa , Humanos , Proteínas Nucleares/genética , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Nuclear/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Latência ViralRESUMO
Reduced expression of DICER, a key enzyme in the miRNA pathway, is frequently associated with aggressive, invasive disease, and poor survival in various malignancies. Regulation of DICER expression is, however, poorly understood. Here, we show that NF90/NF110 facilitates DICER expression by controlling the processing of a miRNA, miR-3173, which is embedded in DICER pre-mRNA. As miR-3173 in turn targets NF90, a feedback amplification loop controlling DICER expression is established. In a nude mouse model, NF90 overexpression reduced proliferation of ovarian cancer cells and significantly reduced tumor size and metastasis, whereas overexpression of miR-3173 dramatically increased metastasis in an NF90- and DICER-dependent manner. Clinically, low NF90 expression and high miR-3173-3p expression were found to be independent prognostic markers of poor survival in a cohort of ovarian carcinoma patients. These findings suggest that, by facilitating DICER expression, NF90 can act as a suppressor of ovarian carcinoma.
Assuntos
Progressão da Doença , Retroalimentação Fisiológica , Proteínas do Fator Nuclear 90/metabolismo , Neoplasias Ovarianas/patologia , Ribonuclease III/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Metástase Neoplásica , Neoplasias Ovarianas/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , Ribonuclease III/genética , Resultado do TratamentoRESUMO
BACKGROUND: Colorectal and breast cancers are among the most common cancers worldwide. They result from a conjugated deficiency of gene maintenance and cell cycle control. OBJECTIVE: We investigate the expression of the microtubule-associated protein MAP9/ASAP and its two partners AURKA and PLK1 in colorectal tumors as well as in ductal breast cancers. MATERIALS AND METHODS: 26 colorectal cancer samples and adjacent normal tissues and 77 ductal breast cancer samples from grade I to grade III were collected. Real-time quantitative PCR was used to analyse the expression of MAP9, AURKA, and PLK1. Results. Expression of MAP9 is downregulated in colorectal cancer compared to normal tissues (P > 10(-3)), whereas those of AURKA and PLK1 are upregulated (P > 10(-4)). In ductal breast cancer, we found a grade-dependent increase of AURKA expression (P > 10(-3)), while the variations of expression of MAP9 and PLK1 are not significant (P > 0.2). CONCLUSIONS: MAP9 downregulation is associated with colorectal malignancy and could be used as a disease marker and a new drug target, while AURKA and PLK1 are upregulated. In ductal breast cancer, AURKA overexpression is strongly associated with the tumor grade and is therefore of prognostic value for the progression of the disease.
Assuntos
Adenocarcinoma/enzimologia , Aurora Quinase A/metabolismo , Neoplasias da Mama/enzimologia , Proteínas de Ciclo Celular/metabolismo , Neoplasias Colorretais/enzimologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Aurora Quinase A/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/patologia , Feminino , Expressão Gênica , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Gradação de Tumores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Quinase 1 Polo-LikeRESUMO
Microtubules are structural components of the cell cytoskeleton and key factors for mitosis and ciliogenesis in eukaryotes. The regulation of MT dynamics requires non-motor MAPs. We previously showed that, in human cells in culture, MAP9 (also named ASAP) is involved in MT dynamics and is essential for mitotic spindle formation and mitosis progression. Indeed, misexpression of MAP9 leads to severe mitotic defects and cell death. Here, we investigated the in vivo role of map9 during zebrafish development. Map9 is expressed mainly as a maternal gene. Within cells, Map9 is associated with the MT network of the mitotic spindle and with centrosomes. Morpholino-mediated depletion of map9 leads to early development arrest before completion of epiboly. Map9 localizes to the MT array of the YSL. This MT network is destroyed in Map9-depleted embryos, and injection of anti-map9 morpholinos directly in the nascent YSL leads to arrest of epiboly/gastrulation. Finally, map9 knockdown deregulates the expression of genes involved in endodermal differentiation, dorso-ventral and left-right patterning, and other MT-based functions. At low morpholino doses, the surviving embryos show dramatic developmental defects, spindle and mitotic defects, and increased apoptosis. Our findings suggest that map9 is a crucial factor in early zebrafish development by regulating different MT-based processes.
Assuntos
Embrião não Mamífero/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Apoptose/genética , Proteínas Associadas aos Microtúbulos/genética , Mitose/fisiologia , Transdução de Sinais , Fuso Acromático/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
p53 is a key tumor suppressor that controls DNA damage response and genomic integrity. In response to genotoxic stress, p53 is stabilized and activated, resulting in controlled activation of genes involved in cell cycle arrest, DNA repair and/or apoptosis. ASAP is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. We show here that following double-strand break DNA formation, ASAP directly interacts with and stabilizes p53 by enhancing its p300-mediated acetylation and blocking its MDM2-mediated ubiquitination and degradation, leading to an increase of p53 transcriptional activity. Upon DNA damage, ASAP is transiently accumulated before being degraded upon persistent damage. This work links the p53 response with the cytoskeleton and confirms that the DNA-damaging signaling pathway is coordinated by centrosomal proteins. We reveal the existence of a new pathway through which ASAP signals the DNA damage response by regulating the p300-MDM2-p53 loop. These results point out ASAP as a possible target for the design of drugs to sensitize radio-resistant tumors.
Assuntos
Dano ao DNA , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proteína p300 Associada a E1A/metabolismo , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/genética , UbiquitinaçãoRESUMO
Bipolar spindle formation is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, abnormal number and structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. ASAP (aster-associated protein or MAP9) is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. Its phosphorylation by Aurora A is required for spindle assembly and mitosis progression. Here, we show that ASAP is localized to the spindle poles by Polo-like kinase 1 (Plk1) (a mitotic kinase that plays an essential role in centrosome regulation and mitotic spindle assembly) through the γ-TuRC-dependent pathway. We also demonstrate that ASAP is a novel substrate of Plk1 phosphorylation and have identified serine 289 as the major phosphorylation site by Plk1 in vivo. ASAP phosphorylated on serine 289 is localized to centrosomes during mitosis, but this phosphorylation is not required for its Plk1-dependent localization at the spindle poles. We show that phosphorylated ASAP on serine 289 contributes to spindle pole stability in a microtubule-dependent manner. These data reveal a novel function of ASAP in centrosome integrity. Our results highlight dual ASAP regulation by Plk1 and further confirm the importance of ASAP for spindle pole organization, bipolar spindle assembly, and mitosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Centrossomo/metabolismo , Imunofluorescência , Humanos , Immunoblotting , Proteínas Associadas aos Microtúbulos/genética , Mitose/genética , Mitose/fisiologia , Fosforilação , Ligação Proteica/genética , Ligação Proteica/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Fuso Acromático/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Quinase 1 Polo-LikeRESUMO
BACKGROUND: ASAP is a newly characterized microtubule-associated protein (MAP) essential for proper cell-cycling. We have previously shown that expression deregulation of human ASAP results in profound defects in mitotic spindle formation and mitotic progression leading to aneuploidy, cytokinesis defects and/or cell death. In the present work we analyze the structure and evolution of the ASAP gene, as well as the domain composition of the encoded protein. Mouse and Xenopus cDNAs were cloned, the tissue expression characterized and the overexpression profile analyzed. RESULTS: Bona fide ASAP orthologs are found in vertebrates with more distantly related potential orthologs in invertebrates. This single-copy gene is conserved in mammals where it maps to syntenic chromosomal regions, but is also clearly identified in bird, fish and frog. The human gene is strongly expressed in brain and testis as a 2.6 Kb transcript encoding a approximately110 KDa protein. The protein contains MAP, MIT-like and THY domains in the C-terminal part indicative of microtubule interaction, while the N-terminal part is more divergent. ASAP is composed of approximately 42% alpha helical structures, and two main coiled-coil regions have been identified. Different sequence features may suggest a role in DNA damage response. As with human ASAP, the mouse and Xenopus proteins localize to the microtubule network in interphase and to the mitotic spindle during mitosis. Overexpression of the mouse protein induces mitotic defects similar to those observed in human. In situ hybridization in testis localized ASAP to the germ cells, whereas in culture neurons ASAP localized to the cell body and growing neurites. CONCLUSION: The conservation of ASAP indicated in our results reflects an essential function in vertebrates. We have cloned the ASAP orthologs in mouse and Xenopus, two valuable models to study the function of ASAP. Tissue expression of ASAP revealed a high expression in brain and testis, two tissues rich in microtubules. ASAP associates to the mitotic spindle and cytoplasmic microtubules, and represents a key factor of mitosis with possible involvement in other cell cycle processes. It may have a role in spermatogenesis and also represents a potential new target for antitumoral drugs. Possible involvement in neuron dynamics also highlights ASAP as a candidate target in neurodegenerative diseases.
Assuntos
Evolução Molecular , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Xenopus/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Clonagem Molecular , Sequência Conservada , DNA Complementar/metabolismo , Expressão Gênica , Humanos , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Células NIH 3T3 , Filogenia , Estrutura Terciária de Proteína , Alinhamento de Sequência , Testículo/metabolismo , Xenopus , Proteínas de Xenopus/análise , Proteínas de Xenopus/metabolismoRESUMO
Proper chromosome segregation is required to maintain the appropriate number of chromosomes from one cell generation to another and to prevent aneuploidy, which is mainly found in solid cancers. A correct mitotic spindle is necessary to accomplish such a process. Aurora kinases play critical roles in chromosome segregation and cell division; their deregulation impairs spindle assembly, checkpoint function and cell division causing chromosome mis-segregation. These kinases have been implicated in tumorigenesis. Aurora-A (AurA), in particular has been identified as a cancer-susceptibility gene, is overexpressed in a number of tumors and is required for G2/M transition and spindle assembly. ASAP is a novel spindle-associated protein, the deregulation of which induces severe mitotic defects. We show here that ASAP is a novel substrate of AurA kinase. We have identified serine 625 as the major phosphorylation site for AurA in vivo and localized the phosphorylated form of ASAP to centrosomes from late G2 to telophase, and around the midbody during cytokinesis. AurA depletion induces a proteasome-dependent degradation of ASAP. ASAP depletion induces spindle defects rescued by the expression of the phosphorylation-mimetic mutant ASAP-S625E and not by the non-phosphorylatable mutant ASAP-S625A. Microinjection of mono-specific S625 phospho-antibodies also impaired spindle formation and mitosis. These results strongly indicate that the phosphorylation of ASAP on S625 by AurA is required for bipolar spindle assembly and is essential for a correct mitotic progression. All together, these results suggest that we have identified a novel AurA substrate, pointing out ASAP as a new potential target for antitumoral drugs.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Aurora Quinases , Linhagem Celular Tumoral , Centrossomo , Citocinese , Humanos , Fosforilação , Serina/metabolismoRESUMO
In mammals, olfaction is mediated by two distinct organs that are located in the nasal cavity: the main olfactory epithelium (MOE) that binds volatile odorants is responsible for the conscious perception of odors, and the vomeronasal organ (VNO) that binds pheromones is responsible for various behavioral and neuroendocrine responses between individuals of a same species. Odorants and pheromones bind to seven transmembrane domain G-protein-coupled receptors that permit signal transduction. These receptors are encoded by large multigene families that evolved in mammal species in function of specific olfactory needs.
Assuntos
Condutos Olfatórios , Feromônios/metabolismo , Receptores Odorantes/genética , Receptores de Feromônios/genética , Animais , Cães , Evolução Molecular , Masculino , Camundongos , Odorantes/análise , Mucosa Olfatória/anatomia & histologia , Primatas , Receptores Odorantes/metabolismo , Olfato , Espermatozoides/metabolismo , Órgão Vomeronasal/anatomia & histologiaRESUMO
We have identified a unique human microtubule-associated protein (MAP) named ASAP for ASter-Associated Protein. ASAP localizes to microtubules in interphase, associates with the mitotic spindle during mitosis, localizes to the central body during cytokinesis and directly binds to purified microtubules by its COOH-terminal domain. Overexpression of ASAP induces profound bundling of cytoplasmic microtubules in interphase cells and aberrant monopolar spindles in mitosis. Depletion of ASAP by RNA interference results in severe mitotic defects: it provokes aberrant mitotic spindle, delays mitotic progression, and leads to defective cytokinesis or cell death. These results suggest a crucial role for ASAP in the organization of the bipolar mitotic spindle, mitosis progression, and cytokinesis and define ASAP as a key factor for proper spindle assembly.
Assuntos
Citocinese/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Sequência de Bases , Western Blotting , Células Cultivadas , Clonagem Molecular , Glutationa Transferase , Humanos , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Oligonucleotídeos , Interferência de RNA , Análise de Sequência de DNA , Fuso Acromático/fisiologiaRESUMO
The sense of smell in mammals can perceive and discriminate a wide variety of volatile odorants. Odorants bind to specific olfactory receptors (ORs) to initiate an action potential that transduces olfactory information to the olfactory cortex. We previously identified the structural motifs of odorant molecules (aliphatic 2- or 3-ketones) required to activate mouse OR912-93 by detection of the odorant response using calcium measurement in transfected cells. In order to study changes in the specificity of this receptor that might have occurred during evolution, we cloned the orthologous genes from six primate species and pig and assayed the encoded receptors for responses to odorants. Primate OR912-93 orthologs share 88-97% sequence identity. All the receptors responded to 2- and 3-heptanone except the squirrel-monkey OR, which responded only to 3-heptanone, and the human and orangutan ORs, which were not functional. Directed mutagenesis allowed us to convert the squirrel-monkey response to that of the other functional 912-93 ORs by substituting three amino acids in the second extracellular loop. Orangutan and human 912-93 ORs regained function after restoration of the arginine residue in the DRY motif required for G-protein activation. However, the human receptor was constitutively activated in the absence of ligand stimulation. Using natural mutants of the OR912-93 receptor, we provide evidence that squirrel-monkeys evolved towards a restriction of the specificity of this receptor and therefore that slight alterations in the sequence of a receptor can induce subtle changes in recognition specificity.
Assuntos
Odorantes , Receptores Odorantes/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos , Animais , Arginina/química , Evolução Biológica , Cálcio/metabolismo , Linhagem Celular , Clonagem Molecular , Evolução Molecular , Humanos , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Filogenia , Pongo pygmaeus/genética , Estrutura Secundária de Proteína , Receptores Odorantes/química , Saimiri , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Suínos , TransfecçãoRESUMO
Antimicrobial peptides provide a defense system against microorganisms. One class of these molecules binds lipophilic substrates and is therefore directed against gram-negative bacteria. This family includes proteins related to bactericidal/permeability-increasing protein (BPI). We characterized an approximately 100-kb cluster of three human genes named RYSR, RYA3, and RY2G5 that are related to the BPI family. The RY cluster maps to 20q11.21, >5 Mb upstream of the BPI cluster. The RY and BPI genes have similar exon structures, indicating that they were derived by duplication from a common ancestor. We identified mouse BPI-related and RY orthologues in syntenic regions, indicating that the gene family expanded before mouse and human diverged. Expression analyses show that RYs are strongly expressed in the olfactory epithelium, suggesting that they also could act as odorant transporters or detoxification agents in the olfactory system. Together, these data show how mammals diversified their antimicrobial defenses/olfactory pathways through a duplication-driven adaptive selection process.
Assuntos
Proteínas Sanguíneas/genética , Cromossomos Humanos Par 20/genética , Evolução Molecular , Duplicação Gênica , Proteínas de Membrana , Família Multigênica/genética , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos , Northern Blotting , Mapeamento Cromossômico , Análise por Conglomerados , Primers do DNA , DNA Complementar/genética , Componentes do Gene/genética , Perfilação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The vomeronasal organ (VNO) is responsible in terrestrial vertebrates for the sensory perception of some pheromones, chemicals that elicit characteristic behaviors among individuals of the same species. Two multigene families (V1R, V2R) that encode proteins with seven putative transmembrane domains that are expressed selectively in different neuron subsets of the VNO have been described in rodents. Pheromone-induced behaviors and a functional VNO have been described in a number of mammals, but this sensory organ seems absent in adult catarrhines and apes, including humans. Until now, only pseudogenes have been isolated in humans, except one putative V1R (hV1RL1) sequence expressed in the main olfactory epithelium. We sought to isolate V1R-like genes in a New World monkey species, the marmoset Callithrix jacchus, that possesses an intact VNO and for which pheromone-induced behavior has been well documented. Using library screening approaches, we have identified five different sequences that exhibit characteristic features of V1R sequences, but that are non-functional pseudogenes. In an attempt to sort out functional V1R genes, we next cloned by polymerase chain reaction (PCR) the primate orthologues of hV1RL1. This approach was successful for gorilla, chimpanzee and orangutan, but not for the other species, including marmoset, probably because these species are too divergent from humans. Chimpanzee and orangutan V1RL1 genes are pseudogenes, whereas the gorilla counterpart is potentially functional. These observations raise the possibility that the V1R family has evolved in such a manner in mammals that every species that relies on a VNO-mediated sensory function possesses its own set of functional vomeronasal genes.
Assuntos
Callithrix/genética , Células Quimiorreceptoras/fisiologia , Fatores Quimiotáticos/genética , Pseudogenes/genética , Órgão Vomeronasal/fisiologia , Sequência de Aminoácidos , Animais , Southern Blotting , Células Quimiorreceptoras/química , Fatores Quimiotáticos/química , Clonagem Molecular , Cosmídeos/genética , Evolução Molecular , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Família Multigênica/genética , Filogenia , Reação em Cadeia da Polimerase , Ratos , Homologia de Sequência de Aminoácidos , Órgão Vomeronasal/químicaRESUMO
The sense of smell is mediated by the initiation of action potential in olfactory sensory neurons during odor stimulation. However, little is known about odorant-olfactory receptor (OR) recognition mechanisms. In the present work, we identified the structural motifs of odorant molecules required to activate mouse OR912-93 by detection of the odorant response using calcium measurement in cells transfected with OR and G(alpha)q and G(alpha)15 proteins. The use of sets of odorants led to the identification of ketones with an aliphatic carbon chain length >or= four carbon atoms and a carbonyl group preferentially located in position C2 or C3. The threshold of detection of these odorants is as low as 10(-6)-10(-8)m. No other odorant ligand, out of 70 representatives of the odorant world, was active. The human ortholog of OR912-93 is not functional, suggesting that apart from a stop-mutation located at the 5'-end that was corrected in the construct, it incurred other deleterious mutations during evolution.