RESUMEN
The telomerase enzyme exists as a large complex (approximately 1,000 kDa) in mammals and at minimum is composed of the telomerase RNA and the catalytic subunit telomerase reverse transcriptase (TERT). In Saccharomyces cerevisiae, telomerase appears to function as an interdependent dimer or multimer in vivo (J. Prescott and E. H. Blackburn, Genes Dev. 11:2790-2800, 1997). However, the requirements for multimerization are not known, and it remained unclear whether telomerase exists as a multimer in other organisms. We show here that human TERT (hTERT) forms a functional multimer in a rabbit reticulocyte lysate reconstitution assay and in human cell extracts. Two separate, catalytically inactive TERT proteins can complement each other in trans to reconstitute catalytic activity. This complementation requires the amino terminus of one hTERT and the reverse transcriptase and C-terminal domains of the second hTERT. The telomerase RNA must associate with only the latter hTERT for reconstitution of telomerase activity to occur. Multimerization of telomerase also facilitates the recognition and elongation of substrates in vitro and in vivo. These data suggest that the catalytic core of human telomerase may exist as a functionally cooperative dimer or multimer in vivo.
Asunto(s)
Telomerasa/química , Catálisis , Proteínas de Unión al ADN , Dimerización , Humanos , Saccharomyces cerevisiae , Relación Estructura-Actividad , Especificidad por Sustrato , Telomerasa/metabolismoRESUMEN
Mammalian telomerase is essential for the maintenance of telomere length [1-5]. Its catalytic core comprises a reverse transcriptase component (TERT) and an RNA component. While the biochemical role of mammalian TERT is well established [6-11], it is unknown whether it is sufficient for telomere-length maintenance, chromosome stability or other cellular processes. Cells from mice in which the mTert gene had been disrupted showed progressive loss of telomere DNA, a phenotype similar to cells in which the gene encoding the telomerase RNA component (mTR) has been disrupted [1,12]. On prolonged growth, mTert-deficient embryonic stem (ES) cells exhibited genomic instability, aneuploidy and telomeric fusions. ES cells heterozygous for the mTert disruption also showed telomere attrition, a phenotype that differs from heterozygous mTR cells [12]. Thus, telomere maintenance in mammals is carried out by a single, limiting TERT.
Asunto(s)
ARN , Telomerasa/fisiología , Telómero/fisiología , Animales , Línea Celular , Proteínas de Unión al ADN , Marcación de Gen , Ratones , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
The minimal, active core of human telomerase is postulated to contain two components, the telomerase RNA hTER and the telomerase reverse transcriptase hTERT. The reconstitution of human telomerase activity in vitro has facilitated the identification of sequences within the telomerase RNA and the RT motifs of hTERT that are essential for telomerase activity. However, the precise role of residues outside the RT domain of hTERT is unknown. Here we have delineated several regions within hTERT that are important for telomerase catalysis, primer use, and interaction with the telomerase RNA and the telomerase-associated protein TEP1. In particular, certain deletions of the amino and carboxy terminus of hTERT that retained an interaction with telomerase RNA and TEP1 were nonetheless completely inactive in vitro and in vivo. Furthermore, hTERT truncations lacking the amino terminus that were competent to bind the telomerase RNA were severely compromised for the ability to elongate telomeric and nontelomeric primers. These results suggest that the interaction of telomerase RNA with hTERT can be functionally uncoupled from polymerization, and that there are regions outside the RT domain of hTERT that are critical for telomerase activity and primer use. These results establish that the human telomerase RT possesses unique polymerization determinants that distinguish it from other RTs.
Asunto(s)
Proteínas Portadoras/metabolismo , ARN/genética , Telomerasa/genética , Telomerasa/metabolismo , Animales , Dominio Catalítico , Línea Celular , Clonación Molecular , Proteínas de Unión al ADN , Células HeLa , Humanos , Riñón , Proteínas de Unión al ARN , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Telomerasa/química , Transcripción Genética , TransfecciónRESUMEN
TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. The mTep1-deficient (mTep1(-/-)) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues from mTep1(-/-) mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1(-/-) mice. Telomere length, even in later generations of mTep1(-/-) mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.
Asunto(s)
Proteínas Portadoras/fisiología , Telómero/fisiología , Animales , Proteínas Portadoras/metabolismo , Catálisis , Embrión de Mamíferos/metabolismo , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Modelos Genéticos , Mutagénesis Sitio-Dirigida , Pruebas de Precipitina , ARN/metabolismo , Proteínas de Unión al ARN , Recombinación Genética , Bazo/citología , Células Madre/metabolismo , Telomerasa , Telómero/ultraestructura , Timo/citologíaRESUMEN
The past few years have brought a flood of new information to the telomerase field. The identification of multiple components of both the telomere and telomerase, the understanding of the importance of telomere maintenance to the long term viability of cells, and the demonstration of the utility of telomerase inhibition in limiting tumor cell growth all convene to provide great enthusiasm for the prospects of targeting the telomerase enzyme in cancer. However, there is clearly much to be learned. Because tumor cells evolve under powerful selection, the emergence of non-telomerase based mechanisms for telomere maintenance should be examined closely. Additionally, the nature of telomerase regulation is currently only poorly understood. More work on the tumor specific regulation of telomerase activity might provide either more opportunities for telomerase inhibition, or more skepticism, as a tumor cell might possess mechanisms for upregulating telomerase activity in the presence of inhibitors. The potential for such regulation has already been observed in certain cell types (46). Currently, the field is intensively investigating the biology and applications of telomere and telomerase biology. In it are great hopes that these fundamental cellular processes might be manipulated to success in the treatment of cancer.
Asunto(s)
Neoplasias/enzimología , Telomerasa/genética , Animales , División Celular , Supervivencia Celular , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Telomerasa/metabolismo , Telómero/fisiologíaRESUMEN
The telomerase catalytic subunit (hTERT) is an essential component of the holoenzyme complex that adds telomeric repeats to the ends of human chromosomes. Maintenance of telomeres by telomerase or another mechanism is required for cell immortalization, and loss of telomeric DNA has been proposed as a trigger for cellular senescence. Available evidence suggests that regulation of telomerase activity primarily depends on transcriptional control of hTERT. However, several human tissues as well as some normal cell strains have been shown to express low levels of hTERT mRNA even though they lack telomerase activity. We have previously identified six splice variants of hTERT, including a "deletion" variant (hTERTalpha) that is missing conserved residues from the catalytic core of the protein. Several of the deletion variants have been detected in normal and developing human tissues. We now show that hTERTalpha inhibits endogenous telomerase activity, which results in telomere shortening and chromosome end-to-end fusions. Telomerase inhibition induced a senescence-like state in HT1080 cells and apoptosis in a jejunal fibroblast cell line. These results suggest a possible role for hTERT splice variants in the regulation of telomerase activity.
Asunto(s)
Empalme Alternativo , Genes Dominantes , ARN , Telomerasa/antagonistas & inhibidores , Telomerasa/genética , Apoptosis , Catálisis , Dominio Catalítico , Línea Celular Transformada/enzimología , Senescencia Celular , Aberraciones Cromosómicas , Cromosomas Humanos/ultraestructura , Proteínas de Unión al ADN , Fibroblastos/citología , Fibroblastos/enzimología , Humanos , Hibridación Fluorescente in Situ , Yeyuno/citología , Mutación Puntual , Subunidades de Proteína , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/fisiología , Eliminación de Secuencia , Telomerasa/química , Telomerasa/fisiología , Telómero/metabolismo , Telómero/ultraestructura , TransfecciónRESUMEN
Vaults are large cytoplasmic ribonucleoprotein complexes of undetermined function. Mammalian vaults have two high molecular mass proteins of 193 and 240 kDa. We have identified a partial cDNA encoding the 240-kDa vault protein and determined it is identical to the mammalian telomerase-associated component, TEP1. TEP1 is the mammalian homolog of the Tetrahymena p80 telomerase protein and has been shown to interact specifically with mammalian telomerase RNA and the catalytic protein subunit hTERT. We show that while TEP1 is a component of the vault particle, vaults have no detectable telomerase activity. Using a yeast three-hybrid assay we demonstrate that several of the human vRNAs interact in a sequence-specific manner with TEP1. The presence of 16 WD40 repeats in the carboxyl terminus of the TEP1 protein is a convenient number for this protein to serve a structural or organizing role in the vault, a particle with eight-fold symmetry. The sharing of the TEP1 protein between vaults and telomerase suggests that TEP1 may play a common role in some aspect of ribonucleoprotein structure, function, or assembly.
Asunto(s)
Proteínas Portadoras/química , Telomerasa/química , Partículas Ribonucleoproteicas en Bóveda/química , Animales , Centrifugación por Gradiente de Densidad , Proteínas de Unión al ADN , Haplorrinos , Células HeLa , Humanos , Ratones , Pruebas de Precipitina , Unión Proteica , ARN/metabolismo , ARN Mensajero/análisis , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Telomerasa/metabolismo , Partículas Ribonucleoproteicas en Bóveda/genética , LevadurasRESUMEN
Despite a strong correlation between telomerase activity and malignancy, the outcome of telomerase inhibition in human tumor cells has not been examined. Here, we have addressed the role of telomerase activity in the proliferation of human tumor and immortal cells by inhibiting TERT function. Inducible dominant-negative mutants of hTERT dramatically reduced the level of endogenous telomerase activity in tumor cell lines. Clones with short telomeres continued to divide, then exhibited an increase in abnormal mitoses followed by massive apoptosis leading to the loss of the entire population. This cell death was telomere-length dependent, as cells with long telomeres were viable but exhibited telomere shortening at a rate similar to that of mortal cells. It appears that telomerase inhibition in cells with short telomeres lead to chromosomal damage, which in turn trigger apoptotic cell death. These results provide the first direct evidence that telomerase is required for the maintenance of human tumor and immortal cell viability, and suggest that tumors with short telomeres may be effectively and rapidly killed following telomerase inhibition.
Asunto(s)
Apoptosis/fisiología , ARN , Telomerasa/metabolismo , Telómero/fisiología , Western Blotting , División Celular , Línea Celular , Proteínas de Unión al ADN , Citometría de Flujo , Genes Dominantes , Humanos , Mitosis , Mutagénesis , Telomerasa/genética , Telómero/genética , Factores de Tiempo , Transfección , Células Tumorales CultivadasRESUMEN
A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27(Kip1) and p21(Cip1). The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G1/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G1, demonstrating that cyclin E2 may be rate limiting for G1 progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catálisis , Ciclo Celular , Línea Celular , Línea Celular Transformada , Clonación Molecular , Quinasa 2 Dependiente de la Ciclina , Ciclinas/genética , ADN Complementario , Fase G1 , Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
Telomerase is a ribonucleoprotein enzyme complex that adds single-stranded telomere DNA to chromosome ends [1]. The RNA component of telomerase contains the template for telomeric DNA addition and is essential for activity [1,2]. Telomerase proteins have been identified in ciliates, yeast and mammals [3-12]. In Saccharomyces cerevisiae, the Est2 protein is homologous to the 123 kDa reverse transcriptase subunit of Euplotes telomerase, and is essential for telomerase activity [8]. In humans, telomerase activity is associated with the telomerase RNA hTR [13], the telomerase RNA-binding protein TP1/TLP1 [5,12] and the TP2 protein encoded by the human EST2 homolog [12] (also known as TRT1, hEST2 or TCS1 [9-11]). The minimal complex sufficient for activity is, however, unknown. We have reconstituted human telomerase activity in reticulocyte lysates and find that only exogenous hTR and TP2 are required for telomerase activity in vitro. Recognition of telomerase RNA by TP2 was species specific, and nucleotides 10-159 of hTR were sufficient for telomerase activity. Telomerase activity immunoprecipitated from the reticulocyte lysate contained hTR and recombinant TP2. Substitution of conserved amino acid residues in the reverse transcriptase domain of TP2 completely abolished telomerase activity. We suggest that TP2 and hTR might represent the minimal catalytic core of human telomerase.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas/metabolismo , ARN no Traducido , ARN/metabolismo , Telomerasa/metabolismo , Catálisis , Sistema Libre de Células , Proteínas de Unión al ADN , Células HeLa , Humanos , Sustancias Macromoleculares , ARN/química , ARN Largo no Codificante , Proteínas de Unión al ARN , Proteínas Recombinantes de Fusión/metabolismo , Reticulocitos , Proteínas de Saccharomyces cerevisiae , Telomerasa/química , Moldes GenéticosRESUMEN
The consequences of long-term in vivo expression of human c-mpl ligand in a mouse model were examined. Transgenic mice expressing the human full-length cDNA in the liver exhibited a fourfold increase in circulating platelet count that persisted stably over the life of the animals. Transgenic animals thrived and appeared healthy for at least 500 days. Transgenic platelets appeared normal with respect to surface antigens and response to platelet aggregation agonists. The highest-expressing transgenic line maintained human c-mpl ligand serum levels of 3 ng/mL. Megakaryocyte numbers in bone marrow and spleen were elevated, as were bone marrow and spleen megakaryocyte colony-forming cells (MEG-CFC). Megakaryocytes were observed in the bone marrow, spleen, liver, and lung, but in no other sites. Circulating myeloid and lymphoid cell populations were increased twofold. Additionally, the animals had a slight but significant anemia despite an increase in marrow colony-forming units-erythroid (CFU-E). No evidence of myelofibrosis was observed in the bone marrow. The platelet nadir in response to administration of either antiplatelet serum (APS) or 5-fluorouracil (5FU) was significantly reduced relative to the control level. Furthermore, the red blood cell (RBC) nadir was reduced relative to control levels in both models, suggesting that c-mpl ligand can directly or indirectly support the maintenance of erythrocyte levels following thrombopoietic insult.
Asunto(s)
Fluorouracilo/administración & dosificación , Inhibidores de Agregación Plaquetaria/administración & dosificación , Trombocitosis/genética , Trombopoyetina/biosíntesis , Animales , ADN Complementario/genética , Expresión Génica , Humanos , Megacariocitos/patología , Ratones , Ratones Transgénicos , Recuento de Plaquetas/efectos de los fármacos , Trombocitosis/patología , Trombocitosis/fisiopatología , Trombopoyetina/genéticaRESUMEN
The telomerase ribonucleoprotein catalyzes the addition of new telomeres onto chromosome ends. A gene encoding a mammalian telomerase homolog called TP1 (telomerase-associated protein 1) was identified and cloned. TP1 exhibited extensive amino acid similarity to the Tetrahymena telomerase protein p80 and was shown to interact specifically with mammalian telomerase RNA. Antiserum to TP1 immunoprecipitated telomerase activity from cell extracts, suggesting that TP1 is associated with telomerase in vivo. The identification of TP1 suggests that telomerase-associated proteins are conserved from ciliates to humans.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , ARN/metabolismo , Telomerasa/química , Secuencia de Aminoácidos , Animales , Northern Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Línea Celular , Clonación Molecular , ADN Complementario/genética , Humanos , Ratones , Datos de Secuencia Molecular , Pruebas de Precipitina , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Homología de Secuencia de Aminoácido , Telomerasa/genética , Telomerasa/metabolismo , Tetrahymena/química , Tetrahymena/genética , Transfección , Células Tumorales CultivadasRESUMEN
We have cloned and characterized a human gene encoding TP2 (telomerase-associated protein 2), a protein with similarity to reverse transcriptases and the catalytic telomerase subunits from Saccharomyces cerevisiae and Euplotes aediculatus. Indirect immunofluorescence revealed that TP2 was localized to the nucleus. Using antibodies to endogenous and epitope-tagged TP2, we found that TP2 was associated specifically with human telomerase activity and the recently identified telomerase-associated protein TP1. Mutation of conserved residues within the reverse transcriptase domain of TP2 severely reduced associated telomerase activity. These results suggest that telomerase is an evolutionarily conserved multisubunit complex composed of both structural and catalytic subunits.
Asunto(s)
Secuencia Conservada , Proteínas/metabolismo , ARN , Telomerasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Catálisis , Núcleo Celular/metabolismo , Proteínas de Unión al ADN , Evolución Molecular , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas/genética , Proteínas de Unión al ARN , ADN Polimerasa Dirigida por ARN/genética , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
The transcription factor E2F-1 plays a central role in the cell cycle through its ability to activate genes involved in cell division. E2F-1 activity is regulated by a number of proteins, including the retinoblastoma susceptibility gene product, cyclin-dependent kinases, and their inhibitors, proteins that have been implicated in the control of certain developmental processes. To investigate a potential role of E2F-1 in differentiation, we assayed the ability of megakaryocytes to form platelets in an in vivo transgenic model. E2F-1 expression in megakaryocytes blocked differentiation during maturation, resulting in severe thrombocytopenia. Ultrastructural analysis of megakaryocytes revealed abnormal development characterized by hyperdemarcation of cytoplasmic membranes and reduced numbers of alpha granules. Administration of megakaryocyte growth and development factor or interleukin 6 could not overcome the differentiation block. Additionally, E2F-1 caused massive megakaryocyte accumulation in both normal and ectopic sites, first evident in E15 embryonic liver. Furthermore, significant apoptosis was observed in transgenic megakaryocytes. These data indicate that E2F-1 can prevent terminal differentiation, probably through its cell cycle-stimulatory activity.
Asunto(s)
Plaquetas/fisiología , Proteínas Portadoras , Proteínas de Ciclo Celular , Ciclo Celular/genética , Proteínas de Unión al ADN , Megacariocitos/fisiología , Factores de Transcripción/genética , Animales , Apoptosis , Secuencia de Bases , Diferenciación Celular , División Celular , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Humanos , Inmunohistoquímica , Hígado/patología , Megacariocitos/patología , Megacariocitos/ultraestructura , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Factor Plaquetario 4/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Proteína 1 de Unión a Retinoblastoma , Bazo/patología , Trombocitopenia/genética , Factor de Transcripción DP1 , Factores de Transcripción/metabolismoRESUMEN
Thrombocytopenia is a condition of multiple etiologies affecting the megakaryocyte lineage. To perturb this lineage in transgenic mice, the tsA58 mutation of the simian virus 40 large tumor antigen was targeted to megakaryocytes using the platelet factor 4 promoter. Ten of 17 transgenic lines generated exhibited low platelet levels, each line displaying a distinct, heritable level of thrombocytopenia. Within a line, the degree of the platelet reduction correlated directly with transgene zygosity. The platelet level could be further reduced by the inactivation of one copy of the endogenous retinoblastoma gene. Western blot analysis detected large tumor antigen protein in the most severely affected lines; less affected lines were below the level of detection. Platelets and megakaryocytes from thrombocytopenic mice exhibited morphological abnormalities. Mice with either normal or reduced platelet levels developed megakaryocytic malignancies with a mean age of onset of about 8 months. There was no correlation between severity of thrombocytopenia and onset of malignancy. These mice provide a defined genetic model for thrombocytopenia, and for megakaryocytic neoplasia, and implicate the retinoblastoma protein in the process of megakaryocyte differentiation.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis , Leucemia Experimental/genética , Megacariocitos/citología , Trombocitopenia/genética , Animales , Secuencia de Bases , Diferenciación Celular , Cartilla de ADN/química , Femenino , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , ARN Mensajero/genética , Proteína de Retinoblastoma/genéticaAsunto(s)
Neoplasias Pulmonares , Vigilancia de la Población , Sistema de Registros , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Neoplasias Pulmonares/epidemiología , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , South Dakota/epidemiología , Tasa de SupervivenciaRESUMEN
Venous access can be a difficult problem for patients receiving intravenous medications, such as chemotherapy, over long periods of time. Totally implanted venous access devices, such as the Port-A-Cath TM (Pharmacia Deltec, St. Paul, MN) have been introduced as a solution to the problem of venous access. These devices are marketed as a safe, reliable method of venous access. Between January 1986 and December 1988, one hundred sixty patients had Port-A-Caths implanted at Sioux Valley Hospital. The medical records of these patients were reviewed to determine the type and rates of complications with the Port-A-Cath. Complications included pneumothoracies, extravasation of injected substances, difficulty aspirating blood, occlusion and sepsis. This article describes the results of our study and a review of the literature.
Asunto(s)
Catéteres de Permanencia/normas , Falla de Equipo/estadística & datos numéricos , Extravasación de Materiales Terapéuticos y Diagnósticos/epidemiología , Infecciones/epidemiología , Neumotórax/epidemiología , Adulto , Catéteres de Permanencia/efectos adversos , Estudios de Evaluación como Asunto , Extravasación de Materiales Terapéuticos y Diagnósticos/etiología , Humanos , Infecciones/etiología , Neumotórax/etiologíaRESUMEN
We describe a technique that uses reverse transcription and the polymerase chain reaction (pcr) to rapidly quantitate numbers of specific mRNA transcripts from nanogram quantities of total cellular RNA. Linearity of input molecules to output signal was maintained by limiting the cycle number and the amount of input RNA and by minimizing the number of manipulations. Absolute levels of specific transcripts were determined by the inclusion of a separate standard curve composed of serially diluted in vitro transcribed RNA run alongside the experimental samples. This allowed rapid quantitation of many samples simultaneously. We applied this technique to measuring the expression of phosphoglycerate kinase 2 (Pgk-2) transgenes in the mouse testis during development. A human PGK-2 transgene, a PGK-2/CAT transgene, and the endogenous mPgk-2 gene all displayed similar patterns and levels of expression, consistent with the conclusion that peak RNA accumulation occurs in pachytene spermatocytes. Mouse protamine 2 (mP2) is expressed at a level approximately tenfold higher than Pgk-2 and displays a different pattern of expression consistent with initiation of transcription occurring in haploid round spermatids.
Asunto(s)
Fosfoglicerato Quinasa/genética , Protaminas/genética , ARN Mensajero/análisis , Espermatogénesis , Espermatozoides/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Espermátides/metabolismo , Espermatocitos/metabolismoRESUMEN
A reverse transcriptase-polymerase chain reaction assay (RT-PCR) was used quantitatively to measure accumulated levels of RNA transcripts in total mouse RNAs derived from male germ cells at various spermatogenic stages. RNA levels for two X-linked enzymes, phosphoglycerate kinase (PGK-1) and hypoxanthine phosphoribosyl transferase (HPRT), both decrease during spermatogenesis, although the transcript levels decrease much more rapidly for PGK-1. RNA for the Y-linked ZFY (zinc finger protein) is elevated in all spermatogenic cell fractions tested, being particularly high in leptotene/zygotene spermatocytes and round spermatids. RNA for adenine phosphoribosyltransferase (APRT) increases 5-fold to a peak during late pachynema. RNA for PGK-2, undetectable in spermatogonial cells, increases at least 50-fold by the round spermatid stage. DNA (cytosine-5-)-methyltransferase (MTase) transcript levels are over an order of magnitude higher throughout spermatogenesis than in non-dividing liver cells.
Asunto(s)
Genes , Espermatogénesis/genética , Transcripción Genética , Adenina Fosforribosiltransferasa/genética , Animales , Secuencia de Bases , ADN (Citosina-5-)-Metiltransferasas/genética , Proteínas de Unión al ADN/genética , Hipoxantina Fosforribosiltransferasa/genética , Masculino , Metaloproteínas/genética , Ratones , Datos de Secuencia Molecular , Fosfoglicerato Quinasa/genética , Reacción en Cadena de la Polimerasa , ARN/análisis , ARN/genética , ZincRESUMEN
The gene encoding testis-specific phosphoglycerate kinase 2 (PGK; ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) is expressed only in meiotic and haploid male germ cells. Transgenic mice containing an 8-kilobase human genomic PGK2 gene express the human gene in a tissue-specific and developmentally regulated manner. To determine the nature and location of sequences controlling this expression, transgenic mice with various lengths of the human PGK2 5' region fused to the chloramphenicol acetyltransferase (CAT) gene were analyzed for expression. A 323-base-pair region 5' to the coding region was found to contain information essential for both tissue-specific and developmentally regulated expression of the CAT reporter gene. Transgenic mice containing a PGK2/luciferase-coding construct were compared with mice containing an equivalent CAT construct. Luciferase gene expression was also testis-specific and was more sensitive than CAT gene expression, but otherwise regulation of the two reporter genes was similar in the germ cells of transgenic mice. Translation of both PGK2/CAT and PGK2/luciferase fusion genes was seen concurrently with the first detectable transcripts.