RESUMEN
Anti-Müllerian hormone (AMH) and its receptor are involved in the regression of Müllerian ducts in male fetuses. We have now cloned and mapped the human AMH receptor gene and provide genetic proof that it is required for AMH signalling, by identifying a mutation in the AMH receptor in a patient with persistent Müllerian duct syndrome. The mutation destroys the invariant dinucleotide at the 5' end of the second intron, generating two abnormal mRNAs, one missing the second exon, required for ligand binding, and the other incorporating the first 12 bases of the second intron. The similar phenotypes observed in AMH-deficient and AMH receptor-deficient individuals indicate that the AMH signalling machinery is remarkably simple, consisting of one ligand and one type II receptor.
Asunto(s)
Trastornos del Desarrollo Sexual/genética , Glicoproteínas , Inhibidores de Crecimiento/fisiología , Conductos Paramesonéfricos/anomalías , Mutación Puntual , Receptores de Péptidos/genética , Hormonas Testiculares/fisiología , Empalme Alternativo , Secuencia de Aminoácidos , Hormona Antimülleriana , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Criptorquidismo/genética , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta , Análisis de Secuencia de ADN , Síndrome , Testículo/química , Transcripción Genética/genéticaRESUMEN
Múllerian Inhibiting Substance (MIS) is a testicular hormone that promotes involution of the Múllerian duct during embryogenesis. The Múllerian duct gives rise to adult female reproductive ducts including the fallopian tubes, uterus, and upper vagina. Thus, testicular MIS ensures the regression of female sex organ primordia. Partially purified bovine MIS was reported to inhibit proliferation of tumor cells derived from human gynecological cancers. These observations suggest that MIS might be an effective anticancer agent for some human tumors. Recombinant human MIS (rHu-MIS) has recently become available. To assess the antiproliferative activity of rHu-MIS, we examined its effects on 11 ovarian, six endometrial, and two nongynecological human tumor cell lines. rHu-MIS had no effect on proliferation of these cell lines in five independent assays. Forty-three primary human tumor explants were also examined in human tumor colony forming assays, gel-supported primary culture assays, and subrenal capsule assays. rHu-MIS significantly inhibited the growth of five of these tumors including four ovarian and one small cell lung cancer explant. The four ovarian cancer responses include three of 13 (23%) explants tested in human tumor colony-forming assays and one of eight (12.5%) explants tested in gel-supported primary culture assays. We conclude that rHu-MIS may have antiproliferative activity against some human ovarian cancers.
Asunto(s)
Neoplasias de los Genitales Femeninos/patología , Glicoproteínas , Inhibidores de Crecimiento , Hormonas Testiculares/farmacología , Animales , Hormona Antimülleriana , División Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Proteínas Recombinantes/farmacología , Ensayo de Capsula Subrrenal , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
GDNFR-alpha is a glycosyl-phosphotidylinositol-linked receptor for glial cell line-derived neurotrophic factor (GDNF). GDNF binds to GDNFR-alpha and this complex, in turn, is believed to interact with the RET receptor tyrosine kinase to effect downstream signalling. GDNFR-alpha belongs to a novel gene family without strong homology to known genes. Thus, little information has been available to help predict genomic structure or location of this gene. In this study, the genomic organization of human GDNFR-alpha was delineated through a combination of PAC clone characterization, long distance PCR and sequence analyses. Exon-intron boundaries were defined by comparing the size and sequence of the genomic PCR products to those predicted by the cDNA sequence. The human GDNFR-alpha gene comprises 9 exons. GDNFR-alpha PAC clones were used for FISH analysis to map this gene to 10q26.
Asunto(s)
Cromosomas Humanos Par 10 , Proteínas de Drosophila , Genoma Humano , Factores de Crecimiento Nervioso , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Secuencia de Bases , Carcinoma Medular/genética , Mapeo Cromosómico , Exones , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Humanos , Hibridación Fluorescente in Situ , Intrones , Proteínas del Tejido Nervioso/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras/metabolismo , Neoplasias de la Tiroides/genética , Células Tumorales CultivadasRESUMEN
Mullerian inhibiting substance (MIS) is a glycoprotein hormone expressed by Sertoli cells that induces the regression of Mullerian ducts during development of the male reproductive tract. Transgenic mice carrying a fusion gene composed of human MIS transcriptional regulatory sequences linked to the SV40 T-antigen gene specifically develop testicular tumors composed of a cell type histologically resembling the Sertoli cell. The lack of pathology at other sites suggests tissue-restricted expression of the transgene. A cell line derived from one of the testicular tumors has been established that continues to express markers associated with Sertoli cells, such as transferrin, sulfated glycoprotein-2, and inhibin-beta B. The cell line does not express detectable levels of inhibin-alpha, MIS, or FSH receptor. However, the cells have retained forskolin responsiveness. As adult Sertoli cells cannot be propagated in vitro, the availability of an immortal cell line displaying features characteristic of normal Sertoli cells should aid in subsequent analyses of the biology of this cell type.
Asunto(s)
Antígenos Transformadores de Poliomavirus/biosíntesis , Genes Sintéticos , Glicoproteínas , Inhibidores de Crecimiento/genética , Oncogenes , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Células de Sertoli/metabolismo , Hormonas Testiculares/genética , Neoplasias Testiculares/genética , Animales , Hormona Antimülleriana , Antígenos Transformadores de Poliomavirus/genética , Línea Celular Transformada , Colforsina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Transgénicos/metabolismo , Especificidad de Órganos , Proteínas Recombinantes de Fusión/genética , Virus 40 de los Simios/genética , Neoplasias Testiculares/patologíaRESUMEN
Mullerian inhibiting substance (MIS)/anti-Mullerian hormone is a differentiation factor that causes regression of the Mullerian duct in the developing male fetus and an apparent sex reversal of the fetal ovary when inappropriately exposed to it. The purified product is a 140-kilodalton glycoprotein composed of two identical subunits. We show that a C-terminal fragment of MIS, which shares homology with transforming growth factor-beta, causes regression of the Mullerian duct and inhibits the biosynthesis of aromatase in the fetal ovary. However, both activities are enhanced dramatically by addition of the N-terminal portion of MIS. Under conditions where potentiation occurs, the N- and C-terminal domains of MIS reassociate. These results indicate that the N-terminus of MIS, unlike that of the other members of the transforming growth factor-beta family, plays a role in maintaining the biological activity of the C-terminus.
Asunto(s)
Glicoproteínas , Inhibidores de Crecimiento/química , Conductos Paramesonéfricos/efectos de los fármacos , Familia de Multigenes , Fragmentos de Péptidos/farmacología , Hormonas Testiculares/química , Factor de Crecimiento Transformador beta , Animales , Hormona Antimülleriana , Anticuerpos Monoclonales/inmunología , Aromatasa/biosíntesis , Células CHO , Bovinos , Cricetinae , Inducción Enzimática/efectos de los fármacos , Femenino , Fibrinolisina/metabolismo , Inhibidores de Crecimiento/inmunología , Inhibidores de Crecimiento/farmacología , Humanos , Ovario/embriología , Ovario/enzimología , Pliegue de Proteína , Ratas , Proteínas Recombinantes/farmacología , Hormonas Testiculares/inmunología , Hormonas Testiculares/farmacologíaRESUMEN
Mullerian inhibiting substance (MIS) is a 140-kilodalton homodimeric glycoprotein that causes regression of the Mullerian ducts in male embryos, and may also have a role in both males and females in the regulation of germ cell maturation. We examined the ontogeny of MIS messenger RNA (mRNA) in rat testes from midgestation through adulthood and found two discrete MIS mRNA species that are developmentally regulated. The larger 2.0-kilobase species is abundant at embryonic day 14, then decreases in late gestation, and is barely detectable after birth. The smaller 1.8-kilobase species is first noted at embryonic day 18 and is the major species detected postnatally. Both species are abundant just prior to birth, at embryonic day 21, then decrease markedly after birth. This variation in MIS mRNA levels correlates with the developmental expression of MIS protein. A series of oligonucleotide-directed ribonuclease H mapping experiments determined that the two mRNA species differ at their 3' ends in the extent of polyadenylation. Thus, differential polyadenylation of MIS mRNA may be an additional mechanism for regulating MIS expression during fetal and postnatal development.
Asunto(s)
Glicoproteínas , Inhibidores de Crecimiento/metabolismo , Poli A/metabolismo , ARN Mensajero/metabolismo , Hormonas Testiculares/metabolismo , Testículo/fisiología , Transcripción Genética , Envejecimiento , Animales , Hormona Antimülleriana , Secuencia de Bases , Sondas de ADN , Femenino , Expresión Génica , Edad Gestacional , Inhibidores de Crecimiento/genética , Masculino , Datos de Secuencia Molecular , Conductos Paramesonéfricos/fisiología , Sondas de Oligonucleótidos , Poli A/genética , Embarazo , ARN Mensajero/genética , Ratas , Ratas Endogámicas , Hormonas Testiculares/genética , Testículo/embriología , Testículo/crecimiento & desarrolloRESUMEN
The ontogeny of expression of anti-Müllerian hormone (AMH) was examined by immunohistochemistry in 135 human gonadal tissue specimens of various developmental age, ranging from 6 weeks of fetal development to 38 yr of postnatal age. The series included specimens from normal testes and ovaries and from individuals either with pathological conditions affecting gonadal development or with idiopathic infertility manifested as azoospermia or severe oligozoospermia. AMH expression was found only in Sertoli and granulosa cells. A 6-week-old fetal testis at the indifferent gonad stage did not yet express AMH. The protein was first visible at 8.5 weeks of development, when sex cords have not yet been formed. Afterward, a majority of testicular specimens, including those from pathological conditions, strongly expressed AMH through fetal development and childhood until puberty. Markedly prolonged expression of AMH was observed in a 20-yr-old 46,XY female with androgen insensitivity syndrome, who retained prepubertal testicular morphology. In normal testes, the switch-off of AMH expression was usually associated with the appearance of primary spermatocytes, suggesting that their presence had an inhibitory effect on AMH. However, in adolescent boys lacking germ cells because of cancer treatment and in a majority of infertile adult men with idiopathic germ cell aplasia, AMH expression was also down-regulated despite the complete lack of spermatogenesis. The decrease in AMH expression thus reflects the terminal differentiation of Sertoli cells and is probably only partially dependent upon a regulatory factor associated with the onset of meiosis. In fetal ovaries, AMH was first detected at 36 weeks gestation in granulosa cells of preantral follicles. Thus, the onset of ovarian expression is at the end of fetal life and not in infancy as previously reported.
Asunto(s)
Envejecimiento/metabolismo , Genitales/metabolismo , Glicoproteínas , Gónadas/embriología , Gónadas/crecimiento & desarrollo , Inhibidores de Crecimiento/metabolismo , Hormonas Testiculares/metabolismo , Adolescente , Adulto , Hormona Antimülleriana , Diferenciación Celular , Niño , Preescolar , Enfermedad , Femenino , Feto/metabolismo , Células de la Granulosa/citología , Humanos , Inmunohistoquímica , Lactante , Masculino , Valores de Referencia , Células de Sertoli/citologíaRESUMEN
Anti-Müllerian hormone (AMH), also called Müllerian inhibiting substance or factor, is produced by Sertoli cells from fetal life until puberty. In the present study, AMH, testosterone (T), LH, and FSH were measured by immunochemical methods in the serum of 50 boys with normal or delayed pubertal development, 4 patients with suspected androgen insensitivity, and 11 patients with either central (CPP) or gonadotropin-independent (GIPP) precocious puberty to investigate the hormonal regulatory mechanisms of AMH secretion at puberty. An inverse relationship between AMH and T levels was demonstrated. In boys with normal or delayed puberty with T concentrations below 6.7 nmol/L, AMH values were elevated (mean +/- SEM, 22.4 +/- 3.1 micrograms/L) and widely dispersed. In subjects with T levels over 6.7 nmol/L, AMH levels were uniformly low (3.4 +/- 0.5 micrograms/L), except in patients with suspected androgen insensitivity. No significant relationship was found between AMH and gonadotropin levels. Similar results were obtained in patients with either CPP or GIPP. Longitudinal studies were performed on four boys with CPP and two with GIPP before and after treatment. At the time of diagnosis, the T concentration was high, and AMH levels were usually low in CPP and GIPP patients alike. When appropriate treatment was initiated, the T concentration was normalized within 2-4 weeks, but restoration of prepubertal AMH levels required several months. Mature Sertoli cells were observed in testicular biopsies performed in three patients with untreated GIPP. Our results suggest that gonadotropins are not directly implicated in repression of AMH synthesis at puberty, but, rather, that the decrease in AMH production is the consequence of an androgen-mediated, long term, reversible chain of events leading to morphological and functional maturation of the Sertoli cells. Thus, the fall in serum AMH levels appears to be an excellent marker of Sertoli cell pubertal development.
Asunto(s)
Glicoproteínas , Inhibidores de Crecimiento/sangre , Pubertad Precoz/sangre , Hormonas Testiculares/sangre , Adolescente , Andrógenos/fisiología , Hormona Antimülleriana , Niño , Resistencia a Medicamentos , Hormona Folículo Estimulante/sangre , Humanos , Hormona Luteinizante/sangre , Masculino , Conductos Paramesonéfricos/fisiología , Pubertad Tardía/sangre , Valores de Referencia , Testosterona/sangreRESUMEN
An enzyme-linked immunosorbent assay has been developed to measure human Müllerian inhibiting substance (MIS) in biological fluids. The enzyme-linked immunosorbent assay is specific for MIS, with a sensitivity in human serum to 0.5 ng/ml and does not recognize transforming growth factor-beta 1 or -beta 2, LH, or FSH. It similarly fails to recognize other proteins secreted from the cell type into which the MIS gene was cloned. MIS was detected in the serum of normal newborns, infants, children, and adults. In males the serum level of MIS is 10-70 ng/mL at birth. The level increases slightly after birth, and then decreases to a basal level of 2-5 ng/mL after the first 10 yr of life. Newborn male urine contains minimal amounts of MIS (0.5 ng/mL). In females MIS is barely detectable in serum at birth, but rises to the basal level equal to that seen in males after 10 yr of age. Similar basal levels of MIS were found in adult ovarian follicular fluid. MIS levels were high in the serum of a female patient with a sex cord tumor (3200 ng/mL), but fell to 100 ng/mL after multiple excisional operations. In addition, a serum MIS level of 20 ng/mL was detected in a patient with an ovarian granulosa cell tumor. A sensitive assay for MIS could be useful in the diagnosis of patients with congenital abnormalities of sexual development and patients with Sertoli cell and/or other MIS-producing neoplasms. Other applications may also be recognized as the biology of MIS in both males and females is further elucidated.
Asunto(s)
Glicoproteínas , Inhibidores de Crecimiento/sangre , Hormonas Testiculares/sangre , Adolescente , Adulto , Factores de Edad , Hormona Antimülleriana , Anticuerpos Monoclonales , Formación de Anticuerpos , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Femenino , Inhibidores de Crecimiento/inmunología , Inhibidores de Crecimiento/uso terapéutico , Humanos , Lactante , Recién Nacido , Masculino , Valores de Referencia , Hormonas Testiculares/inmunología , Hormonas Testiculares/uso terapéuticoRESUMEN
Anti-Müllerian hormone (AMH), also called Müllerian inhibiting substance or factor, is secreted in high amounts by the immature Sertoli cell; it is negatively regulated by testosterone at puberty. In the present study, we measured serum AMH in 20 patients with defects of androgen synthesis or action: 9 with complete androgen insensitivity syndrome, 9 with a partial form, 1 patient with 3 beta-hydroxysteroid dehydrogenase deficiency, and 1 with Leydig cell agenesis. AMH was also determined in 15 control patients with idiopathic male pseudohermaphroditism. The serum AMH concentration was elevated in all testosterone-insensitive or -deficient patients compared with control levels during the first year of life. From 1 yr of age to the onset of puberty, serum AMH levels in patients with androgen insensitivity returned to normal values, but after pubertal development began, AMH levels again rose to extremely high levels in the complete androgen insensitivity syndrome. These results suggest that AMH is negatively regulated by testosterone not only at puberty, but also during the postnatal period. An elevation of serum AMH appears to be an interesting marker of androgen resistance or defect of androgen production in sexually ambiguous male infants.
Asunto(s)
Andrógenos/fisiología , Glicoproteínas , Inhibidores de Crecimiento/sangre , Hormonas Testiculares/sangre , Adolescente , Envejecimiento/sangre , Hormona Antimülleriana , Niño , Preescolar , Trastornos del Desarrollo Sexual/sangre , Resistencia a Medicamentos , Humanos , Lactante , Masculino , Concentración Osmolar , Valores de Referencia , Testosterona/fisiologíaRESUMEN
To determine whether mammalian Müllerian-inhibiting substance (MIS) is active in birds, Müllerian ducts from 7- to 8-day-old male or female chick embryos were cultured in the presence of human recombinant MIS at concentrations between 2.5 and 12.5 micrograms/ml. None of 20 ducts regressed at any concentration. In contrast, at concentrations of 2.5-5 micrograms/ml, all 12 Müllerian ducts from 13-day-old male mouse embryos and 13 out of 14 female ducts were inhibited to varying degrees. It is concluded that avian Müllerian ducts are unresponsive to mammalian MIS. There may be a difference in structure between the MIS of birds and mammals, or the signal-transduction system may be different.
Asunto(s)
Glicoproteínas , Inhibidores de Crecimiento/farmacología , Conductos Paramesonéfricos/efectos de los fármacos , Hormonas Testiculares/farmacología , Animales , Hormona Antimülleriana , Embrión de Pollo , Femenino , Humanos , Masculino , Ratones , Conductos Paramesonéfricos/crecimiento & desarrollo , Técnicas de Cultivo de Órganos , Proteínas Recombinantes/farmacología , Especificidad de la EspecieRESUMEN
The chemiluminescent detection methods described in this chapter have been successfully applied to the detection of plasmid DNA and genomic DNA in Southern and sequencing protocols. The high sensitivity and the simplicity of AMPPD are instrumental in making the chemiluminescent detection of DNA successful in hybridization assays. This detection technique has also been used to detect DNA in dot blots and in situ hybridization experiments as well as proteins in enzyme-linked immunosorbent assays (ELISAs) and Western blots.
Asunto(s)
ADN Recombinante/análisis , ADN/análisis , Sondas de Oligonucleótidos , Adamantano/análogos & derivados , Fosfatasa Alcalina , Autorradiografía/métodos , Secuencia de Bases , Southern Blotting/métodos , ADN/genética , Electroforesis en Gel de Poliacrilamida/métodos , Hibridación in Situ , Indicadores y Reactivos , Mediciones Luminiscentes , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Radioisótopos de Fósforo , Plásmidos , Proteínas/análisisRESUMEN
Since monoclonal antibodies (MAb) specific for CD4 are potent inhibitors of HIV and SIV replication in vitro, we explored their potential usefulness in vivo as an AIDS therapy. The anti-CD4 MAb 5A8 binds to domain 2 of the CD4 molecule and inhibits virus replication and virus-induced cell fusion at a postvirus binding step. Administration of this MAb to normal rhesus monkeys coats all circulating and lymph node CD4 cells and induces neither CD4 cell clearance nor measurable immunosuppression. In the present study, monkeys chronically infected with the simian immunodeficiency virus of macaques (SIVmac) had stable levels of SIVmac provirus in PBMC prior to treatment as measured by a quantitative polymerase chain reaction technique. Six infected monkeys treated with anti-CD4 MAb demonstrated a significant decrease in SIVmac provirus level after 9 days. Of these monkeys, 3 had > 800 CD4 cells/microliter and developed strong antimouse Ig responses that prevented further treatment. The remaining 3 monkeys had < 800 CD4 cell/microliter and failed to develop antimouse Ig antibody responses. When treatment was continued for 12-21 days in these monkeys, a sustained or further decrease in SIVmac provirus load occurred over the extended treatment period. Four monkeys that received a control MAb of irrelevant specificity for 9-22 days showed either no significant change or a transient increase in SIVmac provirus. Thus, the passive administration of anti-CD4 MAb may exert a specific antiviral effect in controlling immunodeficiency virus infection in vivo.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Antígenos CD4/inmunología , Provirus/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/terapia , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Anticuerpos Antiidiotipos/biosíntesis , Secuencia de Bases , Recuento de Linfocito CD4 , Cartilla de ADN/genética , ADN Viral/análisis , ADN Viral/genética , Infecciones por VIH/terapia , Infecciones por VIH/virología , Humanos , Inmunoterapia Adoptiva , Macaca mulatta , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Provirus/genética , Provirus/fisiología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Replicación Viral/inmunologíaRESUMEN
Three acidic arginine esterases have been isolated from the venom of the Western Diamondback rattlesnake (Crotalus atrox). These components demonstrated marked differences in ionic characteristics, as noted by KCl gradient elution from a DEAE-Sephadex A-50 column. Molecular weights, as determined from gel permeation chromatography, were estimated at 25,100 (fraction D), 24,000 (B); and 22,900 (F) for the three separate enzymes. The two larger enzymes (B and D) exhibited similar activities toward the synthetic substrates alpha-N-benzoyl-L-arginine ethyl ester and alpha-N-benzoyl-DL-arginine-p-nitroanilide. Hydrolysis rates were similar to commercial trypsin preparations. Fraction F exhibited a markedly lower activity as an arginine esterase and negligible activity as an arginine amidase. Arginine esterase activity was evident for all three enzymes in the presence of ethylenediamine tetra-acetic acid.
Asunto(s)
Hidrolasas de Éster Carboxílico/aislamiento & purificación , Venenos de Crotálidos/análisis , Animales , Hidrolasas de Éster Carboxílico/farmacología , Caseínas/metabolismo , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Ácido Edético/farmacologíaRESUMEN
We have optimized conditions for genomic Southern analysis of eukaryotic DNA with both unique and degenerate oligonucleotide probes. Using the human Factor IX gene, optimal probe concentrations and hybridization times were determined, and washing conditions with sodium chloride and tetramethylammonium chloride (TMA-Cl) were compared. With TMA-Cl, the washing temperature was independent of GC content. With these conditions, the Factor IX gene was detected in genomic DNA using a 128-fold degenerate 20-mer and a 32-fold degenerate 17-mer. This permits the detection of a gene prior to cloning and the reduction of probe degeneracy, and facilitates the isolation of that gene. We apply this method of probe degeneracy reduction using probes for the human Factor VIII gene.
Asunto(s)
ADN/genética , Genes , Sondas de Oligonucleótidos , Secuencia de Bases , Southern Blotting/métodos , Factor IX/genética , Factor VIII/genética , Variación Genética , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Albúmina Sérica/genéticaRESUMEN
In Bacillus licheniformis, alpha-amylase production varied more than 100-fold depending on the presence or absence of a catabolite-repressing carbon source in the growth medium. alpha-Amylase was produced during the growth phase and not at the onset of the stationary phase. Induction of alpha-amylase correlated with synthesis of mRNA initiating at the promoter of the alpha-amylase gene.
Asunto(s)
Bacillus/enzimología , alfa-Amilasas/biosíntesis , Bacillus/genética , Bacillus/crecimiento & desarrollo , Bacillus/metabolismo , Citratos/metabolismo , Ácido Cítrico , Medios de Cultivo , Inducción Enzimática , Glucosa/metabolismo , Glutamatos/metabolismo , Ácido Glutámico , Regiones Promotoras Genéticas , Almidón/metabolismo , Transcripción Genética , alfa-Amilasas/genéticaRESUMEN
To investigate the role of Müllerian-inhibiting substance (MIS) in mammalian sexual development, we generated MIS-deficient mice. Although MIS-deficient males had testes that were fully descended and produced functional sperm, they also developed female reproductive organs, which interfered with sperm transfer into females, rendering most of these males infertile. Their testes had Leydig cell hyperplasia and, in one instance, neoplasia. The actions of the two primary hormones of male sexual differentiation were genetically eliminated using the testicular feminization (Tfm) mutation in combination with the MIS mutant allele. XY Tfm/MIS double mutants developed as females, with a uterus, coiled oviducts, and no male reproductive organs except undescended dysfunctional testes. These results suggest that eliminating the presumptive female reproductive tract in male fetuses facilitates fertility and that in testes MIS is a negative regulator of Leydig cell proliferation. Eliminating the presumptive male reproductive tract is necessary for proper oviductal morphogenesis during female mouse development.
Asunto(s)
Genitales Masculinos/embriología , Glicoproteínas , Inhibidores de Crecimiento/fisiología , Conductos Paramesonéfricos/embriología , Diferenciación Sexual/fisiología , Hormonas Testiculares/fisiología , Síndrome de Resistencia Androgénica/embriología , Animales , Hormona Antimülleriana , División Celular , Trastornos del Desarrollo Sexual/genética , Trastornos del Desarrollo Sexual/patología , Femenino , Fertilidad/fisiología , Genitales Masculinos/anatomía & histología , Inhibidores de Crecimiento/genética , Células Intersticiales del Testículo/fisiología , Masculino , Ratones , Ratones Noqueados , Morfogénesis , Oviductos/crecimiento & desarrollo , Hormonas Testiculares/genética , Testículo/patología , Conductos Mesonéfricos/embriologíaRESUMEN
We identified two promoters for the beta-lactamase gene of plasmid pBR322. RNA isolated from bacteria containing pBR322 or RNA transcribed in vitro on pBR322 templates was hybridized to 5' end-labeled single-stranded plasmid probes (Berk, A. J., and Sharp, P. A. (1977) Cell 12, 721-732). Electrophoretic analysis of the nuclease S1 digestion products next to Maxam-Gilbert sequencing ladders closely defines the transcriptional initiation points. The natural promoter lies near the coding sequence of the beta-lactamase gene, initiating transcription at -35 bases before the ATG initiation codon, while a second promoter initiates at positions -244 and/or -245 (on the opposite side of the Eco RI site). This promoter overlaps the promoter transcribing in the opposite direction toward the tetracycline gene(s) and starts in the -10 region of that promoter. S1 mapping of procaryotic mRNA, transcribed in vivo, allows both an accurate identification of promoters and the analysis of their transcriptional regulation.
Asunto(s)
Endonucleasas/metabolismo , Escherichia coli/genética , Genes Bacterianos , Operón , ARN Bacteriano/análisis , beta-Lactamasas/genética , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Moldes Genéticos , Transcripción GenéticaRESUMEN
We have investigated whether DNA methylation is involved in regulating the expression of the rat insulin genes. We studied cytosine methylation in and near the two insulin genes to examine the correlation of site-specific methylation with expression in different tissues and to compare the demethylation patterns of the two genes. For both genes, we found certain sites undermethylated only in insulin-producing tissues. However, the overall patterns of the two genes in expressing tissues are quite different. The insulin I gene is significantly less methylated than the insulin II gene in a tumor that makes equal amounts of the two insulin RNAs. In another tumor and cell line that make 5-fold higher levels of insulin I RNA than insulin II RNA, the changes in the methylation levels of the two genes do not correlate with the increased expression of insulin I. In nonexpressing tissues, the insulin I gene is completely methylated, while the insulin II gene is demethylated at a number of sites. Thus, the general level of methylation of the insulin genes does not correlate with their differential expression. Furthermore, the methylation at specific sites does not correlate either; some CG dinucleotides that appear to be important for one gene are not present in the other. Our results suggest that there is no specific control by methylation of the expression of the insulin genes in the rat.
Asunto(s)
ADN/metabolismo , Regulación de la Expresión Génica , Insulina/genética , Animales , Línea Celular , Citosina/metabolismo , Enzimas de Restricción del ADN , Insulinoma/metabolismo , Metilación , Hibridación de Ácido Nucleico , Neoplasias Pancreáticas/metabolismo , ARN/metabolismo , RatasRESUMEN
Lipocortins (annexins) are a family of calcium-dependent phospholipid-binding proteins with phospholipase A2 inhibitory activity. The characteristic primary structure of members of this family consists of a core structure of four or eight repeated domains, which have been implicated in calcium-dependent phospholipid binding. In two lipocortins (I and II) a short amino-terminal sequence distinct from the core structure has potential regulatory functions which are dependent on its phosphorylation state. We have isolated the rat and the human lipocortin I genes and found that they both consist of 13 exons with a striking conservation of their exon-intron structure and their promoter and amino acid sequences. Both lipocortin I genes are at least 19 kbp in length with exons ranging from 57 to 123 bp interrupted by introns as large as 5 kbp. Each of the four repeat units of lipocortin I are encoded by two consecutive exons while individual exons code for the highly conserved putative calcium-binding domains. The promoter sequences in the rat and in human genes are highly conserved and contain nucleotide sequences characterized as enhancer sequences in other genes. The structure of the lipocortin I gene lends support to the hypothesis that the lipocortin genes arose by a duplication of a single domain.