RESUMO
Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.
Assuntos
Cromossomos Artificiais Humanos , Terapia Genética/métodos , Vetores Genéticos , Animais , Linhagem Celular , Cromossomos Humanos Par 21 , Clonagem Molecular , Técnicas de Transferência de Genes , Humanos , Camundongos , Recombinação GenéticaRESUMO
Human-chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.
Assuntos
Cromossomos de Mamíferos/genética , Evolução Molecular , Pan troglodytes/genética , Mapeamento Físico do Cromossomo , Animais , Cromossomos Humanos Par 21/genética , Perfilação da Expressão Gênica , Genes/genética , Genômica , Humanos , Mutagênese/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Retroelementos/genética , Análise de Sequência de DNARESUMO
Circadian rhythms of behavior are driven by oscillators in the brain that are coupled to the environmental light cycle. Circadian rhythms of gene expression occur widely in peripheral organs. It is unclear how these multiple rhythms are coupled together to form a coherent system. To study such coupling, we investigated the effects of cycles of food availability (which exert powerful entraining effects on behavior) on the rhythms of gene expression in the liver, lung, and suprachiasmatic nucleus (SCN). We used a transgenic rat model whose tissues express luciferase in vitro. Although rhythmicity in the SCN remained phase-locked to the light-dark cycle, restricted feeding rapidly entrained the liver, shifting its rhythm by 10 hours within 2 days. Our results demonstrate that feeding cycles can entrain the liver independently of the SCN and the light cycle, and they suggest the need to reexamine the mammalian circadian hierarchy. They also raise the possibility that peripheral circadian oscillators like those in the liver may be coupled to the SCN primarily through rhythmic behavior, such as feeding.
Assuntos
Ritmo Circadiano , Alimentos , Regulação da Expressão Gênica , Fígado/fisiologia , Animais , Animais Geneticamente Modificados , Corticosterona/sangue , Corticosterona/farmacologia , Técnicas de Cultura , Ingestão de Alimentos , Feminino , Genes Reporter , Luciferases/genética , Pulmão/fisiologia , Masculino , Atividade Motora , Especificidade de Órgãos , Ratos , Núcleo Supraquiasmático/fisiologiaRESUMO
In multicellular organisms, circadian oscillators are organized into multitissue systems which function as biological clocks that regulate the activities of the organism in relation to environmental cycles and provide an internal temporal framework. To investigate the organization of a mammalian circadian system, we constructed a transgenic rat line in which luciferase is rhythmically expressed under the control of the mouse Per1 promoter. Light emission from cultured suprachiasmatic nuclei (SCN) of these rats was invariably and robustly rhythmic and persisted for up to 32 days in vitro. Liver, lung, and skeletal muscle also expressed circadian rhythms, which damped after two to seven cycles in vitro. In response to advances and delays of the environmental light cycle, the circadian rhythm of light emission from the SCN shifted more rapidly than did the rhythm of locomotor behavior or the rhythms in peripheral tissues. We hypothesize that a self-sustained circadian pacemaker in the SCN entrains circadian oscillators in the periphery to maintain adaptive phase control, which is temporarily lost following large, abrupt shifts in the environmental light cycle.
Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Ciclo Celular , Técnicas de Cultura , Escuridão , Genes Reporter , Luz , Fígado/fisiologia , Luciferases/genética , Luciferases/metabolismo , Pulmão/fisiologia , Masculino , Camundongos , Atividade Motora , Músculo Esquelético/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteínas Circadianas Period , Regiões Promotoras Genéticas , RatosRESUMO
A Japanese family with atypical type I familial amyloidotic polyneuropathy (FAP) in Iiyama, Japan was studied. Most of the family members have dysfunctions in the central nervous system, in addition to typical symptoms of type I FAP. The transthyretin (TTR, also called prealbumin) gene of the atypical FAP(FAP-IY) was analyzed with recombinant DNA techniques and a RIA method. FAP-IY was found to have the mutation responsible for the methionine-for-valine substitution at position 30 of TTR, as in the case of typical type I FAP. However, analysis of DNA polymorphisms in the TTR locus showed that FAP-IY has a genetic background differing from that of the typical type I FAP. These observations lead to the consideration that a genetic factor(s) involved in the dysfunction of the central nervous system may locate in a chromosome region in close proximity to the TTR gene.
Assuntos
Amiloidose/genética , Polineuropatias/genética , Tratos Piramidais , Degenerações Espinocerebelares/genética , Genes , Humanos , Japão , Mutação , Linhagem , Pré-Albumina/genéticaRESUMO
The FMS-like tyrosine kinase 3 (FLT3) gene, belonging to the receptor tyrosine kinase (TK) subclass III family, plays an important role in normal hematopoiesis and is one of the most frequently mutated genes in hematologic malignancies as well as an attractive target for directed inhibition. Activating mutations of this gene, including internal tandem duplication in the juxtamembrane (JM) domain and point mutations in the TK domain, are found in approximately one-third of patients with acute myeloid leukemia and in a smaller subset of patients with acute lymphoblastic leukemia. We report here that FLT3 may contribute to leukemogenesis in a patient with myeloproliferative disorder and a t(12;13)(p13;q12) translocation through generating a fusion gene with the ETS variant gene 6 (ETV6) gene. ETV6 has been reported to fuse to various partner genes, including TK and transcription factors. Both ETV6/FLT3 and reciprocal FLT3/ETV6 transcripts were detected in the patient mRNA by reverse transcriptase-polymerase chain reaction. At the protein level, however, only ETV6/FLT3 products were expressed. Among them, one retains the helix-loop-helix (HLH) oligomerization domain of ETV6 and the JM as well as TK domain of FLT3. FLT3 receptor in leukemic cells might be inappropriately activated through dimerization by HLH domain of ETV6, which consequently interfered with proliferation and differentiation of hematopoietic cells.
Assuntos
Cromossomos Humanos Par 12 , Cromossomos Humanos Par 13 , Fusão Gênica , Síndrome Hipereosinofílica/genética , Transtornos Mieloproliferativos/genética , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Repressoras/genética , Translocação Genética , Tirosina Quinase 3 Semelhante a fms/genética , Idoso , Clonagem Molecular , Feminino , Humanos , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão/genética , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Stimulation of G protein- or tyrosine kinase-coupled receptors regulates cell proliferation through intracellular Ca(2+) ([Ca(2+)](i)) signaling. In A7r5 cells, we confirmed that inositol 1,4,5-trisphosphate (IP(3)) mediates vasopressin (VP)-evoked Ca(2+) release from intracellular stores and showed that types 1 (IP(3)R(1)) and 3 (IP(3)R(3)) IP(3) receptors were expressed. Using antisera selective for IP(3)R(1) or IP(3)R(3) and another that interacted equally well with both subtypes, together with membranes from SF:9 cells expressing only single IP(3)R subtypes to calibrate immunoblotting, we established that A7r5 cells express 81% IP(3)R(1) and 19% IP(3)R(3). To elucidate the contributions of IP(3)R(1) and IP(3)R(3) to Ca(2+) signaling and proliferation, stable clones expressing promoter-inducible antisense cDNA fragments (-90 to +9) corresponding to the two IP(3)R subtypes were selected. Mild inhibition of IP(3)R(1) (71+/-8% of control level) slightly attenuated the IP(3)-evoked Ca(2+) release (IICR) induced by VP but significantly decreased the subsequent capacitative Ca(2+) entry (CCE) and proliferation. Moderate inhibition (34+/-6%) strongly decreased both IICR and CCE and further blocked proliferation. Complete inhibition almost abolished IICR and CCE and arrested proliferation entirely. Complete inhibition of IP(3)R(3) expression slightly attenuated IICR without affecting CCE or proliferation. In cells microinjected with a low dose of heparin, VP-induced CCE was more susceptible than IICR to mild inhibition of both IP(3)R(1) and IP(3)R(3). A high dose of heparin had a similar effect to complete inhibition of IP(3)R(1) expression: it blocked VP-evoked IICR entirely and CCE by 90%. We conclude that IP(3)R(1), but not IP(3)R(3), is crucial for IICR, CCE, and proliferation of vascular smooth muscle cells.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Músculo Liso Vascular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Relação Dose-Resposta a Droga , Heparina/administração & dosagem , Immunoblotting , Inositol 1,4,5-Trifosfato/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Microinjeções , Músculo Liso Vascular/citologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Vasopressinas/metabolismo , Vasopressinas/farmacologiaRESUMO
A method based on the multiplex polymerase chain reaction (PCR) and gel electrophoresis for the comparative analysis of gene expression levels was developed. Using the method many cDNA fragments from different sources can be compared simultaneously. Competitive PCR amplification of expressed genes from different sources was performed by using 'module-shuffling primers' (MPSs). The MPSs (labeled with different fluorophores) consist of sequence modules of 3 or 4 nt. The modules are arranged in different orders in each primer; therefore, the base sequences of the primers are different but their melting temperatures are identical. The genes expressed in different sources are ligated with tags complementary with the MPSs. Tag-ligated fragments are mixed in one tube and amplified at the same amplification efficiency by the MPSs. Amplified fragments are detected separately by multiple-color gel electrophoresis. This method can detect different amounts of each expressed gene, up to a difference in amounts of 30%, and its detection limit is 0.1 amol per assay.
Assuntos
Cor , Primers do DNA/genética , Perfilação da Expressão Gênica/métodos , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Ligação Competitiva , Calibragem , Linhagem Celular , DNA Complementar/análise , DNA Complementar/química , DNA Complementar/genética , Corantes Fluorescentes/metabolismo , Globinas/genética , Peso Molecular , Desnaturação de Ácido Nucleico , Especificidade de Órgãos , RNA Mensageiro/análise , RNA Mensageiro/química , RNA Mensageiro/genética , Coelhos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Temperatura , TermodinâmicaRESUMO
The involvement of immune response in the resistance of chemically induced stomach cancer was studied in a resistant rat strain (Buffalo) and a sensitive rat strain (ACI). Groups of 10 male Buffalo and ACI rats, 6 weeks of age, were given drinking water with or without N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; 100 mg/l) for 14 days. Total RNA was isolated from the stomach pyloric mucosa from five rats, and cDNA was prepared with reverse transcriptase. Tissue sections of the stomach pyloric mucosa from five rats were stained with antibodies recognizing molecules expressed by various immune cells. Reverse transcription-PCR (RT-PCR), competitive RT-PCR, and Northern blot demonstrated that the expression of MHC class II group genes [MHC class II, MHC class II-associated invariant chain (Ii), CD4 and IgM (B cell marker)], MHC class I group genes (MHC class I and CD8), B7-1 (costimulator on dendritic cells), and CD28 (receptor to B7 on T cells) in the pyloric mucosa was elevated by MNNG in both rat strains but was elevated to a 4-7-fold greater extent in Buffalo rats than in ACI rats. These genes were scarcely expressed in control rats. Histochemical antibody staining after MNNG exposure showed a greater number of cells stained with monoclonal antibody to Ii, OX-62 (dendritic cell marker), and ED-1 (dendritic cell and macrophage common marker) in the interstitial tissue of the pyloric mucosa of Buffalo rats compared with ACI rats. Cell proliferation, as measured by 5-bromo-2-deoxyuridine (BrdUrd)-labeling indices, revealed the presence of BrdUrd-labeled cells only among epithelial cells in the proliferative zone; cells in the interstitial tissue were not labeled with BrdUrd. The results suggest the involvement of dendritic cell response in the resistance to the MNNG induction of stomach carcinogenesis in rats.
Assuntos
Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias Gástricas/imunologia , Animais , Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2 , Northern Blotting , Antígenos CD28/metabolismo , Carcinógenos , Divisão Celular , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/imunologia , Mucosa Gástrica/patologia , Imunidade Celular , Masculino , Glicoproteínas de Membrana/metabolismo , Metilnitronitrosoguanidina , Reação em Cadeia da Polimerase , Piloro/efeitos dos fármacos , Piloro/imunologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos BUF , Especificidade da Espécie , Neoplasias Gástricas/induzido quimicamente , Neoplasias Gástricas/patologiaRESUMO
Administration of N-methyl-N'-nitro-N-nitrosoguanidine, a glandular stomach carcinogen, at the concentration of 100 microg/ml in drinking water for 8 days induced the appearance of a MHC class II-associated invariant chain in the target organ of stomach pyloric mucosa of male Lewis rats. The up-regulation of the MHC class II-associated invariant chain was revealed by fluorescent differential display analysis, reverse transcription-PCR, Northern blot, and histochemical staining. The appearance of MHC class II and MHC class I was also demonstrated by reverse transcription-PCR and Northern blot. The results suggest the involvement of MHC-controlled immune reactions in chemically-induced stomach carcinogenesis.
Assuntos
Antígenos de Histocompatibilidade Classe II/metabolismo , Neoplasias Gástricas/imunologia , Animais , Northern Blotting , Carcinógenos , Clonagem Molecular , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/imunologia , Masculino , Metilnitronitrosoguanidina , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/imunologia , Reação em Cadeia da Polimerase , Piloro , Ratos , Neoplasias Gástricas/induzido quimicamenteRESUMO
Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s) might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have identified for the first time a homozygously deleted region at the marker D1S244 within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines, NB-1 and NB-C201 (MASS-NB-SCH1), although our genotyping has suggested the possibility that both lines are derived from the same origin. The 800-kb PAC contig covering the entire region of homozygous deletion was made and partially sequenced (about 60%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes within the region which include three known genes (DFF45, PGD, and CORT) as well as three other genes which have been reported during processing our present project for the last 3(1/2) years (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14). They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor, although RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes. Oncogene (2000) 19, 4302 - 4307
Assuntos
Cromossomos Humanos Par 1/genética , Regulação Neoplásica da Expressão Gênica , Perda de Heterozigosidade , Neuroblastoma/genética , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Deleção de Sequência , Proteínas de Transporte/genética , Mapeamento Cromossômico , DNA Complementar/genética , Proteínas Fúngicas/genética , Deleção de Genes , Genes , Genes Supressores de Tumor , Marcadores Genéticos , Impressão Genômica , Genótipo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Cinesinas/genética , Proteínas de Membrana/genética , N-Acetilglucosaminiltransferases/genética , Neuroblastoma/patologia , Precursores de Proteínas/genética , Células Tumorais Cultivadas , Enzimas de Conjugação de UbiquitinaRESUMO
A mouse mutation, termed goku, was generated by a gene-trap strategy. goku homozygous mice showed dwarfism, a marked increase in anxiety, and an analgesic effect. Molecular analysis indicated that the mutated gene encodes a puromycin-sensitive aminopeptidase (Psa; EC 3. 4.11.14), whose functions in vivo are unknown. Transcriptional arrest of the Psa gene and a drastic decrease of aminopeptidase activity indicated that the function of Psa is disrupted in homozygous mice. Together with the finding that the Psa gene is strongly expressed in the brain, especially in the striatum and hippocampus, these results suggest that the Psa gene is required for normal growth and the behavior associated with anxiety and pain.
Assuntos
Aminopeptidases/genética , Ansiedade/genética , Nanismo/genética , Dor/genética , Aminopeptidases/deficiência , Aminopeptidases/metabolismo , Animais , Ansiedade/fisiopatologia , Cruzamentos Genéticos , Nanismo/enzimologia , Nanismo/fisiopatologia , Técnicas Genéticas , Vetores Genéticos , Crescimento/genética , Heterozigoto , Homozigoto , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes Neurológicos , Atividade Motora , Dor/fisiopatologia , Desempenho Psicomotor , Transcrição GênicaRESUMO
The mechanisms of amyloid formation in Familial Amyloidotic Polyneuropathy (FAP) are unknown, as well as the factors determining the development of this pathology. To get some insights into this process, we have first tested a fluorimetric assay with thioflavine T, as a quantitative method for transthyretin (TTR) amyloid estimation, using amyloid isolated from post-mortem tissues of a FAP patient. Then production of amyloid fibrils from soluble TTR was achieved by acidification and optimized for protein concentration and pH. The effect of different ions such as metal and sulphate ions in the process of amyloid formation from wild type TTR was compared using a kinetic assay. Under the conditions tested sulphate diminishes the amount of amyloid formed from wild type TTR and in addition appears to promote aggregation of preexisting amyloid fibrils. The relative amyloidogenicity of three TTR variants, TTR Met30, TTR Pro55 and TTR Met119 respectively, was evaluated using a pH dependent assay. It was shown that the Pro55 variant is highly susceptible to amyloid formation as compared to the wild type protein; on the contrary, the Met119 variant is more resistant than the other TTR proteins towards precipitation into amyloid. These results are in agreement with the pathological conditions associated with these mutations. This type of assay has a wide application for testing the influence of other factors, such as therapeutical agents, on amyloid formation.
Assuntos
Neuropatias Amiloides/metabolismo , Amiloide/biossíntese , Pré-Albumina/fisiologia , Neuropatias Amiloides/genética , Humanos , Metais/farmacologia , Microscopia Eletrônica , Pré-Albumina/genéticaRESUMO
Differential display (DD), an arbitrarily primed RT-PCR fingerprinting technique, is a novel approach for the search of differentially expressed transcripts. Using our improved DD protocol, reproducible cDNA fingerprints were successfully obtained from RNAs of Xenopus laevis embryos at six representative stages. Parallel comparison among the fingerprints revealed a number of bands with differential expression patterns. Analysis with clones of three randomly chosen bands confirmed that their expression patterns were faithfully reflected on fingerprints, thereby proving the reliability and validity of the approach. Nucleotide sequencing of these clones revealed that one is identical with a known transcript (cardiac actin), the second is a novel developmentally regulated gene showing no significant homology with those reported previously, and the last is a close but unique relative of XK endo B gene showing somewhat different spatial expression pattern. These results indicated that the DD analysis provides a rapid and reliable way for the identification of novel differentially expressed genes as well as a unique 'scope' for the survey of the changes in overall gene expression profiles occurring in the early embryonic development of Xenopus as well as of other organisms.
Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Xenopus laevis/embriologiaRESUMO
The progress of world-wide efforts in genome mapping and sequencing is uncovering a number of novel genes whose functions cannot be predicted from their structures, thereby demonstrating the need for the systematic collection of biological information other than their sequences to exploit fully the genomic data. Since one of the most fundamental pieces of information is the expression profile of individual genes, various approaches to genome-wide gene-expression scanning are being explored, based on differential hybridization, comparative cDNA sequencing and message-display techniques. Although each approach has unique advantages and drawbacks, the accumulation of expression data on individual genes is expected to shed light on the functions of genes that are revealed by genome analysis but whose function is not known. At the same time, further improvements in these techniques, as well as the development of novel methodologies, are required to explore fully the genome expression status in various biological situations.
Assuntos
Mapeamento Cromossômico/métodos , Expressão Gênica , Técnicas Genéticas , DNA Complementar , Projeto Genoma Humano , Humanos , Hibridização In Situ/métodos , Reação em Cadeia da Polimerase/métodos , RNA/química , RNA/genética , Análise de Sequência de DNA/métodosRESUMO
Maternal recognition of pregnancy in rodents requires semicircadian surges of hypophyseal PRL secretion during early gestation, which are required for the formation of the corpus luteum of pregnancy (CLP). Here we show that puromycin-sensitive aminopeptidase (Psa)-deficient mice display female infertility that results from impaired formation of CLP. Transplantation of mutant ovaries into normal females restored fertility but not vice versa. Psa-deficient females revealed no semicircadian surges of PRL induced after mating stimuli. Pregnancy in the mutant females was restored by grafting intact pituitaries to elevate circulating levels of PRL. Psa is thus required for the appearance of the semicircadian surges of PRL secretion that are crucial for maintaining pregnancy in rodents.
Assuntos
Aminopeptidases/metabolismo , Corpo Lúteo/fisiologia , Estro/fisiologia , Prenhez , Prolactina/metabolismo , Aminopeptidases/deficiência , Aminopeptidases/genética , Animais , Células Cultivadas , Ritmo Circadiano , Dopamina/farmacologia , Implantação do Embrião , Feminino , Immunoblotting , Infertilidade Feminina , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovário/transplante , Hipófise/citologia , Gravidez , Prolactina/administração & dosagem , Prolactina/genética , Útero/anatomia & histologiaRESUMO
Male reproductive performance is composed of two principal elements, copulation and spermatogenesis. A wealth of literature has described the intricate web of endocrine events underlying these biological processes. In the present study we show that puromycin-sensitive aminopeptidase (Psa)-deficient mice are infertile, lack copulatory behavior, and have impaired spermatogenesis. The reproductive deficits of the mutants are not restored by androgen administration, although no aberrant localization of the sex steroid receptors was detectable in their brains and testes. Considering the strong expression of the Psa gene in the brain and Sertoli cells and the degenerative morphology of Sertoli cells in Psa-deficient mice, Psa may participate in testosterone-mediated reproductive signal pathways in the brain and testis.
Assuntos
Aminopeptidases/metabolismo , Infertilidade Masculina/enzimologia , Comportamento Sexual Animal , Espermatogênese , Testículo/fisiologia , Aminopeptidases/deficiência , Aminopeptidases/genética , Animais , Feminino , Citometria de Fluxo , Hormônio Foliculoestimulante/farmacologia , Genótipo , Hipotálamo/citologia , Hipotálamo/metabolismo , Immunoblotting , Imuno-Histoquímica , Infertilidade Masculina/genética , Inibinas/metabolismo , Hormônio Luteinizante/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Prolactina/farmacologia , Receptores Androgênicos/metabolismo , Receptores de Estrogênio/metabolismo , Glândulas Seminais/citologia , Glândulas Seminais/efeitos dos fármacos , Túbulos Seminíferos/citologia , Túbulos Seminíferos/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Testículo/metabolismo , Testículo/ultraestrutura , Testosterona/farmacologiaRESUMO
We previously described a case of familial hyperproinsulinemia, the fifth to be reported. In the present study we characterized the genetic defect carried by this family and demonstrated that it could be detected by polymerase chain reaction-single stranded conformational polymorphism. Since the serum proinsulin molecule from the propositus, a 63-yr-old Japanese man, was eluted on the same fraction of human proinsulin intermediate cleaved only at the B-C junction, we sequenced exon 3 of his insulin gene, including the C-A junction. A point mutation was discovered that changed codon 65 from arginine (CGT) to histidine (CAT) in one allele. This was the same point mutation as that described previously in three unrelated kindreds representing two races, consistent with the hypothesis that the dinucleotide sequence CpG may be a "hot spot" for mutations. Recently, developed polymerase chain reaction-single stranded conformational polymorphism proved useful in detecting this mutation in the family members. The daughter of the propositus and one of his two grandsons were also demonstrated to be heterozygous for this point mutation by this method.
Assuntos
DNA de Cadeia Simples/genética , Insulina/genética , Mutação Puntual , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético , Proinsulina/sangue , Sequência de Bases , Eletroforese , Éxons , Humanos , Masculino , Pessoa de Meia-Idade , Conformação Molecular , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , LinhagemRESUMO
Down syndrome is the most common birth defect, which is caused by trisomy 21. We identified a novel gene in the so-called Down syndrome critical region by EST mapping to genomic DNA and following cDNA cloning. The gene, designated DCRB (Down syndrome Critical Region gene B), consisted of three exons of 1095 bp in total and encoded a large open reading frame of 118 amino acid residues. The amino acids sequence of DCRB showed no significant homology to any known protein. Northern blot analysis showed that DCRB is mainly expressed in the placenta, in which a major 1.1-kb band and a minor 2.0-kb band were detected. Minor bands of 1.4 kb and 2.2 kb were also detected in adult heart and skeletal muscle.
Assuntos
Aberrações Cromossômicas/genética , Cromossomos Humanos Par 21/genética , DNA Complementar/química , Síndrome de Down/genética , Placenta/química , Proteínas da Gravidez/genética , Homologia de Sequência do Ácido Nucleico , Adulto , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Química Encefálica/genética , Transtornos Cromossômicos , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Feminino , Expressão Gênica , Humanos , Lactente , Fígado/química , Masculino , Dados de Sequência Molecular , Miocárdio/química , Reação em Cadeia da Polimerase , Gravidez , Proteínas da Gravidez/química , Proteínas da Gravidez/isolamento & purificação , RNA Longo não Codificante , RNA Mensageiro/análise , RNA Mensageiro/química , Transcrição GênicaRESUMO
From human chromosome 21-specific libraries, 22 SfiI linking clones and 38 P1 clones were isolated and regionally mapped on the chromosome. The terminal sequences of these clones were determined and pairs of PCR primers were generated which could specifically amplify the sequenced regions. These sequence-tagged sites (STSs) should be useful for constructing a high resolution map of human chromosome 21.