RESUMEN
Tonically active cholinergic interneurons (TANs) from the nucleus accumbens (NAc) are centrally involved in reward behavior. TANs express a vesicular glutamate transporter referred to as VGLUT3 and thus use both acetylcholine and glutamate as neurotransmitters. The respective roles of each transmitter in the regulation of reward and addiction are still unknown. In this study, we showed that disruption of the gene that encodes VGLUT3 (Slc17a8) markedly increased cocaine self-administration in mice. Concomitantly, the amount of dopamine (DA) release was strongly augmented in the NAc of VGLUT3(-/-) mice because of a lack of signaling by metabotropic glutamate receptors. Furthermore, dendritic spines and glutamatergic synaptic transmission on medium spiny neurons were increased in the NAc of VGLUT3(-/-) mice. Increased DA and glutamate signaling in the NAc are hallmarks of addiction. Our study shows that TANs use glutamate to reduce DA release and decrease reinforcing properties of cocaine in mice. Interestingly, we also observed an increased frequency of rare variations in SLC17A8 in a cohort of severe drug abusers compared with controls. Our findings identify VGLUT3 as an unexpected regulator of drug abuse.
Asunto(s)
Trastornos Relacionados con Cocaína/genética , Trastornos Relacionados con Cocaína/patología , Dopamina/metabolismo , Predisposición Genética a la Enfermedad/genética , Ácido Glutámico/metabolismo , Núcleo Accumbens/metabolismo , Transducción de Señal/fisiología , Proteínas de Transporte Vesicular de Glutamato/genética , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Adulto , Animales , Cocaína/farmacología , Condicionamiento Operante/efectos de los fármacos , Inhibidores de Captación de Dopamina/farmacología , Humanos , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Núcleo Accumbens/citología , Núcleo Accumbens/efectos de los fármacos , Trastornos Relacionados con Opioides/genética , Trastornos Relacionados con Opioides/patología , Autoadministración , Potenciales Sinápticos/efectos de los fármacos , Potenciales Sinápticos/genética , Proteínas de Transporte Vesicular de Glutamato/deficienciaRESUMEN
DNA studies of the human genome have shown polymorphic variation at thousands of sites, defining an absolute genetic uniqueness for each individual. There are many circumstances in which it may be desirable to diagnose this molecular individuality, as for instance, in criminal investigations or paternity testing. Several techniques can be used for this DNA diagnosis and we can choose among them the one that best suits the specific problem at hand. In this review we describe the main methodologies in current use to investigate human DNA polymorphisms, discussing the best application of each option, as well as their advantages and disadvantages.
Asunto(s)
Dermatoglifia del ADN , Variación Genética , Genoma Humano , Polimorfismo Genético , Bioética , Ligamiento Genético , Marcadores Genéticos , Genética Médica , Humanos , Fenotipo , Polimorfismo de Longitud del Fragmento de Restricción , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Acetylcholine is one of the major modulators of brain functions and it is the main neurotransmitter at the peripheral nervous system. Modulation of acetylcholine release is crucial for nervous system function. Moreover, dysfunction of cholinergic transmission has been linked to a number of pathological conditions. In this manuscript, we review the cellular mechanisms involved with regulation of acetylcholine synthesis and storage. We focus on how phosphorylation of key cholinergic proteins can participate in the physiological regulation of cholinergic nerve-endings.
Asunto(s)
Acetilcolina/metabolismo , Proteínas de Transporte de Membrana , Proteínas de Transporte Vesicular , Acetilcolina/biosíntesis , Animales , Proteínas Portadoras/metabolismo , Colina O-Acetiltransferasa/metabolismo , Humanos , Vesículas Sinápticas/enzimología , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular de AcetilcolinaRESUMEN
4-Aminobenzovesamicol was used to test whether activation of protein kinase C protects the vesicular acetylcholine transporter from interaction with vesamicol-like drugs. The essentially irreversible vesamicol analog inhibits the release of newly synthesized [3H]acetylcholine from stimulated hippocampal slices. Prior activation of protein kinase C with a phorbol ester prevented the inhibition of [3H]acetylcholine release, but activation of protein kinase C after the exposure to the irreversible analog did not prevent the effect of the drug. Binding of 4-aminobenzovesamicol in hippocampal synaptosomes, assayed using [3H]vesamicol and back-titration, was decreased by activation of protein kinase C prior to analog exposure but not by activation subsequent to exposure. We propose that phosphorylation of the vesicular acetylcholine transporter prevents the binding of vesamicol-like drugs.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Transporte de Membrana , Piperidinas/metabolismo , Proteínas de Transporte Vesicular , Acetilcolina/antagonistas & inhibidores , Acetilcolina/biosíntesis , Animales , Estimulación Eléctrica , Activación Enzimática/fisiología , Femenino , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Piperidinas/farmacología , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Proteínas de Transporte Vesicular de AcetilcolinaRESUMEN
The present experiments investigated the release of [3H]acetylcholine ([3H]ACh) from the guinea pig myenteric plexus treated with 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a drug that impairs ACh accumulation by synaptic vesicles. Ouabain, an Na+-K+ ATPase inhibitor, released [3H]ACh synthesised in the presence of (-)-vesamicol, while electrical field stimulation or KCl depolarisation were not effective to release the transmitter in this condition. The effect of ouabain was Ca2+-dependent and in the presence of (-)-vesamicol it was blocked by calphostin C, an inhibitor of protein kinase C (PKC). In addition, stimulation of kinase C activity by a phorbol ester, but not by its inactive isomer, prevented (-)-vesamicol from interfering with the release of [3H]ACh in electrically-stimulated myenteric plexus, similar to the effect of ouabain. We conclude that release of [3H]ACh induced by ouabain in the presence of (-)-vesamicol depends on PKC activation.
Asunto(s)
Acetilcolina/metabolismo , Plexo Mientérico/fisiología , Fármacos Neuromusculares Despolarizantes/farmacología , Piperidinas/farmacología , Proteína Quinasa C/metabolismo , Animales , Calcio/metabolismo , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Femenino , Cobayas , Cinética , Masculino , Plexo Mientérico/efectos de los fármacos , Naftalenos/farmacología , Ouabaína/farmacología , Cloruro de Potasio/farmacología , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/fisiología , TritioRESUMEN
A non-toxic protein (TsNTxP) from Tityus serrulatus scorpion venom has been shown to be an efficient immunogen and anti-TsNTxP antibodies recognize and neutralize the effect of Tityus serrulatus venom [Chávez-Olórtegui et al., 1997. Toxicon 35, 213-221]. With the purpose of studying the organization of the gene that code for this protein, we have isolated a full length cDNA clone for TsNTxP from a cDNA expression library using anti-TsNTxP antibodies. The nucleotide sequence of the gene that encodes TsNTxP was also obtained and it reveals the presence of an intron within the signal peptide sequence. The TsNTxP gene showed high degree of similarity with genes encoding toxins from scorpions of the genus Tityrus.
Asunto(s)
Venenos de Escorpión/química , Venenos de Escorpión/genética , Animales , Anticuerpos , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Intrones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Señales de Clasificación de Proteína/química , Venenos de Escorpión/inmunología , Análisis de Secuencia de ADNRESUMEN
From a Phoneutria nigriventer venom gland cDNA library several clones coding for the insect specific neurotoxin Tx4(6-1) were isolated. cDNA analysis showed that the encoded protein contained three distinct segments, comprising a signal sequence of 16 amino acids, followed by a glutamate-rich sequence of 18 amino acids and, finally, the coding region for the mature toxin. The deduced amino acid sequence for the mature polypeptide was identical to the protein sequence determined chemically. In addition, two new putative toxins called Pn4A and Pn4B were characterized and their predicted complete amino acid sequence revealed approximately 78% similarity to Tx4(6-1).
Asunto(s)
ADN Complementario/genética , Insecticidas , Neurotoxinas/genética , Péptidos/genética , Venenos de Araña/genética , Arañas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/aislamiento & purificación , Glándulas Exocrinas/química , Glándulas Exocrinas/metabolismo , Datos de Secuencia Molecular , Neurotoxinas/química , Venenos de Araña/químicaRESUMEN
PnTx3-1 is a peptide isolated from the venom of the spider Phoneutria nigriventer that specifically inhibits A-type K(+) currents (I(A)) in GH(3) cells. Here we used a bacterial expression system to produce an NH(2)-extended mutant of PnTx3-1 (ISEF-PnTx3-1) and tested whether the toxin is functional. The recombinant toxin was purified from bacterial extracts by a combination of affinity and ion-exchange chromatography. The recombinant toxin blocked A-type K(+) currents in GH(3) cells in a fashion similar to that observed with the wild-type toxin purified from the spider venom. These results suggest that recombinant cDNA methods provide a novel source for the production of functional Phoneutria toxins. The recombinant ISEF-PnTx3-1 should be useful for further understanding of the role of A-type K(+) currents in biological processes.
Asunto(s)
Neuropéptidos/biosíntesis , Bloqueadores de los Canales de Potasio , Proteínas Recombinantes de Fusión/biosíntesis , Venenos de Araña/genética , Secuencia de Aminoácidos , Animales , Fraccionamiento Químico , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Clonación Molecular , Datos de Secuencia Molecular , Neuropéptidos/genética , Neuropéptidos/farmacología , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Venenos de Araña/farmacología , Células Tumorales CultivadasRESUMEN
A cDNA library made from venom glands of the spider Phoneutria nigriventer was constructed and used to clone neurotoxic peptides. A cDNA of about 360 nucleotides encoding the precursor for the toxin Tx2-1 active on mammals has been isolated. The deduced amino acid sequence for the mature polypeptide confirms the polypeptide sequence previously published. In addition, two new putative toxins called Pn2-1A and Pn2-5A have been characterized and their complete amino acid sequence show 92% similarity to Tx2-1 and 94% similarity to Tx2-5 respectively. The cDNAs revealed that the precursors contain signal peptides characterized by a very hydrophobic core and a propeptide interposed between the signal sequence and the peptide toxin.
Asunto(s)
Clonación Molecular/métodos , ADN Complementario/química , Biblioteca de Genes , Neurotoxinas/química , Venenos de Araña/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/química , Proteínas Recombinantes , ArañasRESUMEN
The cDNAs (Tx3-2 and Pn3A) encoding precursor of toxin Tx3-2 and an isoform called Pn3A have been isolated from a library constructed from stimulated venom glands of the spider Phoneutria nigriventer. The cDNA of Tx3-2 reveals the presence of a signal peptide of 21 amino acids and of an intervening propeptide (with 16 amino acids) preceding the toxin sequence, which was followed by additional amino acid residues at the C-terminus (C-terminal peptide), implying post-translational modifications of the synthesised peptide. The deduced amino acid sequence for the mature toxin confirms the previous sequence published. In addition, by using the whole-cell patch clamp technique, we have determined that purified Tx3-2 decreases L-type currents present in GH3 cells. Finally, the presence of the cDNA Pn3A, with high sequence identity with Tx3-2, reveals the existence of a putative new toxin showing, at the cDNA level, 85.4% identity in its whole segment.
Asunto(s)
Canales de Calcio/efectos de los fármacos , Péptidos/genética , Venenos de Araña/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación de Organismos , ADN Complementario/química , Biblioteca de Genes , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Señales de Clasificación de Proteína , Venenos de Araña/clasificación , Venenos de Araña/toxicidadRESUMEN
1. Human amniotic fluid contains a complex mixture of proteins, of which only the minority are of fetal origin. We have identified the fetal polypeptides of second trimester amniotic fluid samples by two different methods. 2. The first method was the side by side comparison of silver-stained two-dimensional polyacrylamide gels of amniotic fluid polypeptides with pregnant female plasma polypeptides, after passage of both through a Blue Sepharose affinity column to remove albumin. The second method was the identification of the fetal polypeptides in two-dimensional Western blots with an antiserum made specific for fetal proteins. 3. Using these techniques we have identified 13 major fetal polypeptide fractions with apparent molecular weights of 220, 200, 82, 70, 59, 52, 50, 36, 30, 25, 20, 18 and 11 kDa. Five of these polypeptides, with molecular weights of 82, 59, 50, 20 and 18 kDa, have not previously identified. The identification of these fetal components provides a reference base for molecular studies of normal and pathological fetal development.
Asunto(s)
Líquido Amniótico/química , Proteínas Fetales/análisis , Péptidos/análisis , Embarazo/sangre , Western Blotting , Electroforesis en Gel de Poliacrilamida/métodos , Femenino , Humanos , Péptidos/sangre , Segundo Trimestre del Embarazo , Albúmina Sérica/aislamiento & purificaciónRESUMEN
T-type Ca2+ channels are important for cell signaling by a variety of cells. We report here the electrophysiological and molecular characteristics of the whole-cell Ca2+ current in GH3 clonal pituitary cells. The current inactivation at 0 mV was described by a single exponential function with a time constant of 18.32 +/- 1.87 ms (N = 16). The I-V relationship measured with Ca2+ as a charge carrier was shifted to the left when we applied a conditioning pre-pulse of up to -120 mV, indicating that a low voltage-activated current may be present in GH3 cells. Transient currents were first activated at -50 mV and peaked around -20 mV. The half-maximal voltage activation and the slope factors for the two conditions are -35.02 +/- 2.4 and 6.7 +/- 0.3 mV (pre-pulse of -120 mV, N = 15), and -27.0 +/- 0.97 and 7.5 +/- 0.7 mV (pre-pulse of -40 mV, N = 9). The 8-mV shift in the activation mid-point was statistically significant (P < 0.05). The tail currents decayed bi-exponentially suggesting two different T-type Ca2+ channel populations. RT-PCR revealed the presence of alpha1G (CaV3.1) and alpha1I (CaV3.3) T-type Ca2+ channel mRNA transcripts.
Asunto(s)
Canales de Calcio Tipo T/fisiología , Hipófisis/citología , Canales de Calcio Tipo T/genética , Línea Celular , Células Clonales , Electrofisiología , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Impaired cholinergic neurotransmission can affect memory formation and influence sleep-wake cycles (SWC). In the present study, we describe the SWC in mice with a deficient vesicular acetylcholine transporter (VAChT) system, previously characterized as presenting reduced acetylcholine release and cognitive and behavioral dysfunctions. Continuous, chronic ECoG and EMG recordings were used to evaluate the SWC pattern during light and dark phases in VAChT knockdown heterozygous (VAChT-KDHET, n=7) and wild-type (WT, n=7) mice. SWC were evaluated for sleep efficiency, total amount and mean duration of slow-wave, intermediate and paradoxical sleep, as well as the number of awakenings from sleep. After recording SWC, contextual fear-conditioning tests were used as an acetylcholine-dependent learning paradigm. The results showed that sleep efficiency in VAChT-KDHET animals was similar to that of WT mice, but that the SWC was more fragmented. Fragmentation was characterized by an increase in the number of awakenings, mainly during intermediate sleep. VAChT-KDHET animals performed poorly in the contextual fear-conditioning paradigm (mean freezing time: 34.4±3.1 and 44.5±3.3 s for WT and VAChT-KDHET animals, respectively), which was followed by a 45% reduction in the number of paradoxical sleep episodes after the training session. Taken together, the results show that reduced cholinergic transmission led to sleep fragmentation and learning impairment. We discuss the results on the basis of cholinergic plasticity and its relevance to sleep homeostasis. We suggest that VAChT-KDHET mice could be a useful model to test cholinergic drugs used to treat sleep dysfunction in neurodegenerative disorders.
Asunto(s)
Conducta Animal/fisiología , Colinérgicos/metabolismo , Aprendizaje por Laberinto/fisiología , Fases del Sueño/fisiología , Transmisión Sináptica/fisiología , Vigilia/fisiología , Animales , Masculino , Ratones , Ratones Noqueados , Modelos AnimalesRESUMEN
PnTx3-4 is a toxin isolated from the venom of the spider Phoneutria nigriventer that blocks N-, P/Q-, and R-type voltage-gated calcium channels and has great potential for clinical applications. In this report we used the SUMO system to express large amounts of recombinant PnTx3-4 peptide, which was found in both soluble and insoluble fractions of bacterial extracts. We purified the recombinant toxin from both fractions and showed that the recombinant peptide showed biological activity similar to the native PnTx3-4. In silico analysis of the primary sequence of PnTx3-4 indicated that the peptide conforms to all the criteria of a knottin scaffold. Additionally, circular dichroism spectrum analysis of the recombinant PnTx3-4 predicted that the toxin structure is composed of approximately 53% turns/unordered, 31% α-helix and 16% ß-strand, which is consistent with predicted model of the PnTx3-4 knottin scaffold available at the knottin database (http://knottin.cbs.cnrs.fr). These studies provide the basis for future large scale production and structure-function investigation of PnTx3-4.
Asunto(s)
Canales de Calcio/metabolismo , Neuropéptidos/metabolismo , Proteínas Recombinantes/metabolismo , Venenos de Araña/metabolismo , Secuencia de Aminoácidos , Análisis de Varianza , Animales , Dicroismo Circular , Datos de Secuencia Molecular , Neuropéptidos/genética , Neuropéptidos/aislamiento & purificación , Oligonucleótidos/genética , Plásmidos/genética , Pliegue de Proteína , Análisis de Secuencia de ADN , Sinaptosomas/metabolismoRESUMEN
The neurotransmitter acetylcholine (ACh) plays a crucial role in both the central and peripheral nervous system. Central cholinergic transmission is important for cognitive functions and cholinergic disruptions have been associated with different neural disorders. We here tested the role of cholinergic transmission in basic cognitive functions, i.e. in prepulse inhibition (PPI) and short-term habituation (STH) as well as long-term habituation (LTH) of startle using mice with a 65% knockdown (KD) of the vesicular ACh transporter (VAChT). These mice are slow in refilling cholinergic synaptic transmitter vesicles, leading to a reduced cholinergic tone. Prepulse inhibition has been assumed to be mediated by cholinergic projections from the midbrain to the reticular formation. Surprisingly, PPI and STH were normal in these mice, whereas LTH was disrupted. This disruption could be rescued by pre-testing injections of the ACh esterase inhibitor galantamine, but not by post-testing injections. The lack of a PPI deficit might be because of the fact that VAChT KD mice show disruptions mainly in prolonged cholinergic activity, therefore the transient activation by prepulse processing might not be sufficient to deplete synaptic vesicles. The disruption of LTH indicates that the latter depends on a tonic cholinergic inhibition. Future experiments will address which cholinergic cell group is responsible for this effect.
Asunto(s)
Acetilcolina/metabolismo , Habituación Psicofisiológica/genética , Filtrado Sensorial/genética , Proteínas de Transporte Vesicular de Acetilcolina/genética , Estimulación Acústica , Animales , Ratones , Ratones Noqueados , Reflejo de Sobresalto/genética , Transmisión Sináptica/genética , Vesículas Sinápticas/genética , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular de Acetilcolina/metabolismoRESUMEN
Storage of acetylcholine in synaptic vesicles plays a key role in maintaining cholinergic function. Here we used mice with a targeted mutation in the vesicular acetylcholine transporter (VAChT) gene that reduces transporter expression by 40% to investigate cognitive processing under conditions of VAChT deficiency. Motor skill learning in the rotarod revealed that VAChT mutant mice were slower to learn this task, but once they reached maximum performance they were indistinguishable from wild-type mice. Interestingly, motor skill performance maintenance after 10 days was unaffected in these mutant mice. We also tested whether reduced VAChT levels affected learning in an object recognition memory task. We found that VAChT mutant mice presented a deficit in memory encoding necessary for the temporal order version of the object recognition memory, but showed no alteration in spatial working memory, or spatial memory in general when tested in the Morris water maze test. The memory deficit in object recognition memory observed in VAChT mutant mice could be reversed by cholinesterase inhibitors, suggesting that learning deficits caused by reduced VAChT expression can be ameliorated by restoring ACh levels in the synapse. These data indicate an important role for cholinergic tone in motor learning and object recognition memory.
Asunto(s)
Discapacidades para el Aprendizaje/genética , Proteínas de Transporte Vesicular de Acetilcolina/biosíntesis , Proteínas de Transporte Vesicular de Acetilcolina/genética , Animales , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente , Discapacidades para el Aprendizaje/psicología , Aprendizaje por Laberinto/fisiología , Recuerdo Mental/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Actividad Motora/fisiología , Destreza Motora/fisiología , Terminaciones Nerviosas/metabolismo , Equilibrio Postural/fisiología , Desempeño Psicomotor/fisiología , Reconocimiento en Psicología/fisiologíaRESUMEN
Impaired cholinergic neurotransmission can affect memory formation and influence sleep-wake cycles (SWC). In the present study, we describe the SWC in mice with a deficient vesicular acetylcholine transporter (VAChT) system, previously characterized as presenting reduced acetylcholine release and cognitive and behavioral dysfunctions. Continuous, chronic ECoG and EMG recordings were used to evaluate the SWC pattern during light and dark phases in VAChT knockdown heterozygous (VAChT-KDHET, n=7) and wild-type (WT, n=7) mice. SWC were evaluated for sleep efficiency, total amount and mean duration of slow-wave, intermediate and paradoxical sleep, as well as the number of awakenings from sleep. After recording SWC, contextual fear-conditioning tests were used as an acetylcholine-dependent learning paradigm. The results showed that sleep efficiency in VAChT-KDHET animals was similar to that of WT mice, but that the SWC was more fragmented. Fragmentation was characterized by an increase in the number of awakenings, mainly during intermediate sleep. VAChT-KDHET animals performed poorly in the contextual fear-conditioning paradigm (mean freezing time: 34.4±3.1 and 44.5±3.3 s for WT and VAChT-KDHET animals, respectively), which was followed by a 45% reduction in the number of paradoxical sleep episodes after the training session. Taken together, the results show that reduced cholinergic transmission led to sleep fragmentation and learning impairment. We discuss the results on the basis of cholinergic plasticity and its relevance to sleep homeostasis. We suggest that VAChT-KDHET mice could be a useful model to test cholinergic drugs used to treat sleep dysfunction in neurodegenerative disorders.
Asunto(s)
Animales , Masculino , Ratones , Conducta Animal/fisiología , Colinérgicos/metabolismo , Aprendizaje por Laberinto/fisiología , Fases del Sueño/fisiología , Transmisión Sináptica/fisiología , Vigilia/fisiología , Ratones Noqueados , Modelos AnimalesRESUMEN
The mouse Y chromosome is rich in repetitive sequences. We describe a new highly male-specific BALB/c mouse sequence named 142-5. The distribution of 142-5 related sequences, which appeared to be repeated at least 100 times in the male genome of Mus musculus, was visualized on the Y chromosome by in situ hybridization. Their accumulation patterns in the genus Mus showed that the sequences evolved quickly and suggested that they might prove useful for detecting genetic differences between closely related species. To test this hypothesis, we isolated 20 additional sequences from three mouse species (M. musculus, M. spicilegus, and M. spretus) and compared their nucleotide sequences using three different computer programs. It was found that the sequences were remarkably similar but could be divided into four subgroups, and that each species had a distinct set or sets of sequences that were amplified in the Y chromosome.
Asunto(s)
Muridae/genética , Secuencias Repetitivas de Ácidos Nucleicos , Cromosoma Y , Animales , Southern Blotting , Femenino , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos BALB C , Especificidad de la EspecieRESUMEN
The strong suppression of crossing-over between the X and Y chromosomes permits rapid accumulation of repetitive sequences in the Y chromosome. To gain insight into the mechanism responsible for the sequence amplification, it is essential to characterize Y chromosomal repetitive sequences at the molecular level. Here, we report the entire nucleotide sequence (3,902bp) of AC11, a mouse sequence that is repeated 300 times in the Y chromosome. AC11 is AT rich (32.8% GC), and contains many short poly(A) sequences. In addition, it has Bkm and LINE sequences as well as a Y chromosome-specific sequence. The Bkm sequence consists of typical (GATA) and (GACA) repeating units, whereas the LINE sequence deviates considerably from other mouse LINE sequences (71-76% identity) and may be considered atypical. The Y chromosome-specific region seems to be unique and does not identify similar sequences in the GenBank library. The information obtained from the nucleotide sequence should form the foundation to study the evolutionary processes through which AC11-related sequences have accumulated in the mouse Y chromosome.
Asunto(s)
ADN/genética , Secuencias Repetitivas de Ácidos Nucleicos , Cromosoma Y , Animales , Secuencia de Bases , Evolución Biológica , Intercambio Genético , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Poli A/genéticaRESUMEN
145SC5 is a Y chromosomal repetitive sequence isolated from a BALB/c mouse and is unique in that it detects poly(A)-containing transcripts in the testis. We determined nucleotide sequences of 145SC5 and a related cDNA clone designated PC11 and compared their sequences to that of pYMT2/B, another related cDNA clone. 145SC5 and PC11 are almost identical (97%), while PC11 and pYMT2/B share 84% identity. In situ hybridization and Southern blot analysis with XXSxr male mice demonstrated that 145SC5-related sequences were distributed over the entire length of the Y chromosome including the short arm. Two types of Y chromosome are present in inbred mouse strains: the Mus musculus musculus type and M.m. domesticus type. Among 26 inbred strains surveyed, 145SC5 detected polymorphims only in the M.m. domesticus Y chromosome.