RESUMO
The trigeminal ganglion (TG) and mesencephalic trigeminal tract nucleus (Mes5) were investigated in wild type and dystonia musculorum (dt) mice to study the effect of dystonin deficiency on primary sensory neurons in the trigeminal nervous system. At postnatal day 14, the number of TG neurons was markedly decreased in dt mice when compared to wild type mice (43.1% reduction). In addition, dystonin disruption decreased the number of sensory neurons which bound to isolectin B4, and contained calcitonin gene-related peptide or high-affinity nerve growth factor receptor TrkA. Immunohistochemistry for caspase-3 demonstrated that dystonin deficiency induced excess cell death of TG neurons during the early postnatal period. In contrast, Mes5 neurons were barely affected in dt mice. These data together suggest that dystonin is necessary for survival of nociceptors but not proprioceptors in the trigeminal nervous system.
Assuntos
Proteínas do Citoesqueleto/deficiência , Proteínas do Tecido Nervoso/deficiência , Nociceptores/metabolismo , Células Receptoras Sensoriais/metabolismo , Gânglio Trigeminal/citologia , Núcleos do Trigêmeo/citologia , Animais , Proteínas de Transporte , Caspase 3/metabolismo , Distonina , Regulação da Expressão Gênica/genética , Lectinas/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Receptor trkA/metabolismoRESUMO
Proximal spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron gene (SMN1). In humans, two nearly identical copies of SMN exist and differ only by a single non-polymorphic C-->T nucleotide transition in exon 7. SMN1 contains a 'C' nucleotide at the +6 position of exon 7 and produces primarily full-length SMN transcripts, whereas SMN2 contains a 'T' nucleotide and produces high levels of a transcript that lacks exon 7 and a low level of full-length SMN transcripts. All SMA patients lack a functional SMN1 gene but retain at least one copy of SMN2, suggesting that the low level of full-length protein produced from SMN2 is sufficient for all cell types except motor neurons. The murine Smn gene is not duplicated or alternatively spliced. It resembles SMN1 in that the critical exon 7 +6 'C' nucleotide is conserved. We have generated Smn minigenes containing either wild-type Smn exon 7 or an altered exon 7 containing the C-->T nucleotide transition to mimic SMN2. When expressed in cultured cells or transgenic mice, the wild-type minigene produced only full-length transcripts whereas the modified minigene alternatively spliced exon 7. Furthermore, Smn exon 7 contains a critical AG-rich exonic splice enhancer sequence (ESE) analogous to the human ESE within SMN exon 7, and subtle mutations within the mESE caused a variation in Smn transcript levels. In summary, we show for the first time that the murine Smn locus can be induced to alternatively splice exon 7. These results demonstrate that SMN protein levels can be varied in the mouse by the introduction of specific mutations at the endogenous Smn locus and thereby lay the foundation for developing animals that closely 'resemble' SMA patients.
Assuntos
Processamento Alternativo , Éxons/genética , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Animais , Composição de Bases/genética , Sequência de Bases , Células COS , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Elementos Facilitadores Genéticos/genética , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/metabolismo , Plasmídeos/genética , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas do Complexo SMN , Homologia de Sequência do Ácido Nucleico , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio Motor , Distribuição Tecidual , Transcrição Gênica , Células Tumorais CultivadasRESUMO
cAMP and dexamethasone are known to modulate Na+ transport in epithelial cells. We investigated whether dibutyryl cAMP (DBcAMP) and dexamethasone modulate the mRNA expression of two key elements of the Na+ transport system in isolated rat alveolar epithelial cells: alpha-, beta-, and gamma-subunits of the epithelial Na+ channel (ENaC) and the alpha1- and beta1-subunits of Na+-K+-ATPase. The cells were treated for up to 48 h with DBcAMP or dexamethasone to assess their long-term impact on the steady-state level of ENaC and Na+-K+-ATPase mRNA. DBcAMP induced a twofold transient increase of alpha-ENaC and alpha1-Na+-K+-ATPase mRNA that peaked after 8 h of treatment. It also upregulated beta- and gamma-ENaC mRNA but not beta1-Na+-K+-ATPase mRNA. Dexamethasone augmented alpha-ENaC mRNA expression 4.4-fold in cells treated for 24 h and also upregulated beta- and gamma-ENaC mRNA. There was a 1.6-fold increase at 8 h of beta1-Na+-K+-ATPase mRNA but no significant modulation of alpha1-Na+-K+-ATPase mRNA expression. Because DBcAMP and dexamethasone did not increase the stability of alpha-ENaC mRNA, we cloned 3.2 kb of the 5' sequences flanking the mouse alpha-ENaC gene to study the impact of DBcAMP and dexamethasone on alpha-ENaC promoter activity. The promoter was able to drive basal expression of the chloramphenicol acetyltransferase (CAT) reporter gene in A549 cells. Dexamethasone increased the activity of the promoter by a factor of 5.9. To complete the study, the physiological effects of DBcAMP and dexamethasone were investigated by measuring transepithelial current in treated and control cells. DBcAMP and dexamethasone modulated transepithelial current with a time course reminiscent of the profile observed for alpha-ENaC mRNA expression. DBcAMP had a greater impact on transepithelial current (2.5-fold increase at 8 h) than dexamethasone (1.8-fold increase at 24 h). These results suggest that modulation of alpha-ENaC and Na+-K+-ATPase gene expression is one of the mechanisms that regulates Na+ transport in alveolar epithelial cells.
Assuntos
AMP Cíclico/farmacologia , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Isoenzimas/metabolismo , Alvéolos Pulmonares/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Bucladesina/farmacologia , Células Cultivadas , Clonagem Molecular , Combinação de Medicamentos , Condutividade Elétrica , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Canais Epiteliais de Sódio , Isoenzimas/genética , Masculino , Regiões Promotoras Genéticas/fisiologia , Biossíntese de Proteínas/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/fisiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Canais de Sódio/genética , Canais de Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Transcrição Gênica/fisiologiaRESUMO
We have isolated from mouse intestine a full-length cDNA clone that encodes an 86-amino acid precursor protein containing a 26-amino acid signal sequence. As deduced from its sequence, the mature 60-aa protein named MPGC60 belongs to the Kazal type of secreted trypsin inhibitors. The MPGC60 peptide has 58% homology with the PEC-60 peptide isolated from pig intestine. In the gut of adult mice, an increasing rostrocaudal gradient in MPGC60 mRNA levels was observed by Northern analysis. In situ hybridization analysis demonstrated strong Mpgc60 expression in Paneth cells and in a subset of goblet cells in the differentiated gut. During postnatal differentiation of the gut, a strong increase in Mpgc60 expression was detected in both small and large intestine. However, in small intestine activation of the Mpgc60 gene occurred earlier than in the large intestine. Apart from the intestinal tract, MPGC60 mRNA was also detectable in the mesenchyme surrounding the uterine epithelium and in endothelia of some blood vessels. However, in contrast to the situation observed in pig, no Mpgc60 expression was detectable by Northern, in situ and reverse transcriptase polymerase chain reaction (RT-PCR) analysis in cells of the immune system, that is, in monocytes, macrophages, peripheral blood and in spleen. Northern blot analysis on mRNA isolated from porcine and murine intestine showed a single transcript in mouse, but several transcripts in pig. Southern blot and fluorescent in situ hybridisation (FISH) analysis demonstrated the presence of a single gene situated in band A of chromosome 4. This region is syntenic with human chromosome regions 6q, 8q and 9p. The gene responsible for human hereditary mixed polyposis syndrome has been localized to human 6q. This raises the possibility that Mpgc60 is a candidate gene for this human disorder.
Assuntos
Regulação da Expressão Gênica/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Genoma , Mucosa Intestinal/metabolismo , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , SuínosRESUMO
Sodium reabsorption by the amiloride-sensitive sodium channel of epithelial cells plays a crucial role in the management of ionic composition and fluid volume in the body. In the respiratory system, sodium transport is involved in the clearance of pulmonary edema and of liquid secreted during fetal life at birth. We have cloned a partial cDNA of the alpha subunit of the mouse amiloride-sensitive sodium channel (alpha mENaC). In the region of comparison, the mouse alpha subunit shows 92% identity at the DNA level and 95% identity at the amino acid level with the rat sequence. The kidneys, lungs, and distal colon are major sites of expression of a 3.5-kb alpha mENaC mRNA. During mouse development, alpha mENaC transcripts appear late during gestation (d 17.5) and are expressed continuously thereafter. In the distal colon, a short 1.2-kb mRNA deleted of the 5' part of the transcript is detected during gestation and is replaced gradually by the mature 3.5-kb transcript after birth. Alpha mENaC and alpha1 Na+-K+-ATPase mRNAs have an expression profile that is modulated similarly during development for a given tissue. The expression of alpha mENaC transcripts increases transiently in the lungs at birth (2.5-fold), as for alpha1 Na+-K+-ATPase mRNAs (1.5-fold), suggesting that the expression of several components of the sodium transport system is modulated in the lungs at that time. In the kidney, there is no significant increase of alpha mENaC and alpha1 Na+-K+-ATPase mRNAs in newborns.
Assuntos
Animais Recém-Nascidos/genética , Feto/química , Canais de Sódio , Canais de Sódio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Colo/química , Colo/fisiologia , Sondas de DNA/genética , Canais Epiteliais de Sódio , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Idade Gestacional , Rim/química , Rim/fisiologia , Pulmão/química , Pulmão/fisiologia , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Gravidez , RNA Mensageiro/análise , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Canais de Sódio/química , Canais de Sódio/fisiologia , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/fisiologia , Transcrição Gênica/genética , Transcrição Gênica/fisiologiaRESUMO
Retinoic acid has been shown to be an anticancer agent, and a growing literature suggests that it is the nuclear retinoic acid receptor beta2 (RARbeta2) that is primarily responsible for mediating this effect, at least in some systems. To determine whether partial inactivation of RARbeta2 would predispose to lung cancer in mice, we generated three transgenic lines expressing antisense sequences. When killed at 13-3/4-18 months of age, 21/36 animals had a total of 43 pulmonary tumors superficially visible upon necropsy, whereas among 23 nontransgenic mice, only 1 had a single visible lung tumor. A twofold higher incidence of lung tumors was seen in homozygous vs. hemizygous antisense mice. The endogenous RARbeta2 message level was reduced in transgenic lung tissue and further reduced in the tumors. RARbeta4, a truncated isoform derived from the same transcript as RARbeta2, does not carry the sequence identified by the antisense construct and its message was not as strongly affected. Immunofluorescence studies showed that RARbeta was virtually undetectable in the tumors, but present in normal tissue. We conclude that RARbeta2, but probably not RARbeta4, plays an important role in suppression of murine lung tumorigenesis.
Assuntos
Genes Supressores de Tumor/genética , Neoplasias Pulmonares/genética , RNA Antissenso/genética , Receptores do Ácido Retinoico/genética , Animais , Sequência de Bases , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Neoplásico/análise , Receptores do Ácido Retinoico/biossíntese , Distribuição TecidualRESUMO
The beta-globin locus control region (LCR) confers high levels of position-independent, copy number-dependent expression onto globin transgenes. Here > 40 independent transgenic mouse lines and founders that carried the LCR in cis with the beta-globin gene promoter driving a lacZ reporter gene were studied. Expression of the lacZ transgene was assayed by measuring beta-galactosidase enzyme activity in fetal liver extracts, the levels of which correlated with the quantity of lacZ mRNA determined using RNase protection assays. Unexpectedly, expression of the lacZ transgene was found to show strong position effects, varying as much as 700-fold per transgene copy. These position effects occurred even if the whole beta-globin gene was incorporated as part of the lacZ reporter gene. Moreover, DNase I-hypersensitive sites appeared in the transgene LCR in high expressing but not in low expressing lines, suggesting that the LCR itself was position dependent. In contrast, MEL cell clones, in which transcriptionally active integration sites were selected for, gave < 13-fold variation in expression per copy of an LCR-lacZ construct. These results show that the lacZ reporter affects the ability of the LCR to activate chromatin in mice and that culture cells are not an adequate model for position-independent gene expression studies.
Assuntos
Globinas/genética , Sequências Reguladoras de Ácido Nucleico , Animais , Resistência a Medicamentos , Genes Reporter , Óperon Lac , Camundongos , Camundongos Transgênicos/embriologia , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The cytosolic chaperonin TRiC is a large protein complex involved in the folding of newly synthesized actin and tubulin. The fertilization of the mouse oocyte is followed by a remodelling of the actin and tubulin filaments. The TRiC subunit TCP1 is expressed only from the 4-cell stage on, even though actin and tubulin are synthesized in the previous stages. We investigated the onset of synthesis of another subunit, TRiC-P5, during early mouse embryogenesis. We report that TRiC-P5 is synthesized at the 2-cell stage in an alpha-amanitin sensitive manner. Thus, it is expressed before TCP1 and is one of the first proteins to be synthesized after zygotic genome activation.
Assuntos
Blastocisto/metabolismo , Chaperoninas/biossíntese , Regulação da Expressão Gênica , Biossíntese de Proteínas , Proteínas , Zigoto/metabolismo , Amanitinas/farmacologia , Animais , Linfoma de Burkitt , Linhagem Celular , Chaperonina com TCP-1 , Citosol/metabolismo , Desenvolvimento Embrionário e Fetal , Feminino , Fertilização , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Superovulação , Células Tumorais CultivadasRESUMO
Transgenic mice were generated which express a truncated nuclear retinoic acid receptor beta (RAR beta), closely resembling the natural isoform RAR beta 4, under the control of the MMTV promoter. The transgene was expressed in salivary gland, testis, lung and mammary tissue in two different lines. At approximately 11-14 months virtually all the transgenic mice showed hyperplasia of the lung alveolar epithelium with an excess of type II pneumocytes. Hyperplasia of the mammary alveoli and terminal ducts was also seen in some females. Salivary glands and some sebaceous glands were hyperplastic in most male transgenic mice, but only rarely in females or in non-transgenics. Primary benign and malignant tumours were more numerous in transgenic mice than in controls, with a total of 23 in 43 mice versus two in 33 non-transgenic animals. Treatment with dexamethasone to increase transgene expression resulted in exaggerated versions of the above phenotypes. Overexpression of RAR beta 4 therefore appears to predispose various tissues to hyperplasia and neoplasia, and this by contrast to the RAR beta 2 isoform, which has tumour suppressor activity. A survey of ratios of RAR beta 4:RAR beta 2 expression in human lung tumour cell lines showed an increase compared with normal lung tissue, suggesting that RAR beta 4 may play a similar role in human tumorigenesis.
Assuntos
Neoplasias Pulmonares/genética , Neoplasias Mamárias Experimentais/genética , Receptores do Ácido Retinoico/genética , Animais , Sequência de Bases , Primers do DNA , Feminino , Regulação da Expressão Gênica , Humanos , Hiperplasia , Incidência , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/epidemiologia , Masculino , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/epidemiologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Glândulas Salivares/metabolismo , Glândulas Salivares/patologia , Glândulas Sebáceas/metabolismo , Glândulas Sebáceas/patologia , Células Tumorais CultivadasRESUMO
We have reinvestigated the neural crest defect of Splotch (Sp1H) mutant embryos using the tissue specific expression of lacZ by the HCMV-IEP-lacZ (CMZ) transgene as a marker. The CMZ transgene was backcrossed onto the Sp1H mutant background, which has been shown to carry mutations in the Pax-3 gene. The CMZ transgene has previously been shown to be expressed in some neural crest-derived neural tissues of midgestation embryos. The pattern of CMZ expression in Splotch mutants is not caused by alterations of transgene transcription, but demonstrates morphological deviations of neural crest development. The gradual size reduction of spinal ganglia along a rostrocaudal gradient is shown to occur concomitantly with a size reduction of the sympathetic ganglia. CMZ expression also reveals the total absence of sympathetic ganglion cells in thoracic and lumbar segments of Sp1H homozygotes, which is confirmed in serial sections. Observations in whole mounts of CMZ transgenic homozygotes suggest that cranial nerve ganglia develop normally in these embryos. CMZ is expressed in epithelial cells around the neural tube defect in Splotch mutants at the epidermal/neuroepithelial boundary. It is proposed that this expression represents premigratory neural crest cells that remain within the epithelial layer around the neural tube defect. These observations are discussed with reference to the normal pattern of Pax-3 expression.
Assuntos
Defeitos do Tubo Neural/embriologia , Fatores de Transcrição , beta-Galactosidase/genética , Animais , Moléculas de Adesão Celular Neuronais/análise , DNA/genética , DNA/isolamento & purificação , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Defeitos do Tubo Neural/patologia , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Gravidez , Sistema Nervoso Simpático/embriologia , beta-Galactosidase/análiseRESUMO
Homozygosity for the Splotch mutation causes neural tube and neural crest defects in mice. It has been demonstrated that Splotch mutant mice carry mutations in the homeodomain of the Pax-3 gene. Pax-3 is expressed in the neural tube, some neural crest derivatives, the mesenchyme of the limb bud and the somites. We have examined the development of the somite-derived skeletal muscles in homozygotes carrying the Splotch (Sp1H) mutation. Our results suggest that the Splotch mutation affects the development of skeletal muscles in a region-specific way: 1. The expression of the CMZ transgene in homozygotes reveals a disorganisation of the dermomyotome in whole stained embryos. 2. The axial musculature is reduced in size along a rostro-caudal gradient. 3. The muscle anlagen in the limbs develop much more slowly. Muscles of the head and the ventral body wall are normally developed in the mutant on day 13.5 of gestation. Recently, it has been shown that the myogenic precursors of the limbs are derived from the lateral half of the somite. The specific disturbance of muscle development in the limbs of Splotch mutants thus suggests a role for Pax-3 in the organisation of the somite, the production of trophic factors in the limb mesenchyme or an alteration of myogenic and mesenchymal cells.
Assuntos
Desenvolvimento Embrionário e Fetal , Extremidades/embriologia , Camundongos Mutantes Neurológicos/embriologia , Músculos/embriologia , Mutação , Animais , Homozigoto , Camundongos , Camundongos Mutantes Neurológicos/genética , Defeitos do Tubo Neural/genéticaRESUMO
The ability of parthenogenetic (pg) cells to contribute to proliferating stem cell populations of postnatal aggregation chimeras was investigated. Using DNA in situ analysis, pg participation was observed in highly regenerative epithelia of various regions of the gastrointestinal tract, e.g., stomach, duodenum and colon, in the epithelia of tongue and uterus and in the epidermis. Pg cells also contributed to the epithelium of the urinary bladder, which is characterized by a relatively slow cellular turnover. Using a sensitive proliferation marker to determine division rate of pg and normal (wt) cells in tissues of a 24-day-old chimera, no significant differences between pg and fertilized cells were observed. However, in colon and uterus of a pg <==> wt chimera aged 101 days, a significant loss of proliferative capacity of pg cells was found. In the colon, this loss of proliferative potential was accompanied by an altered morphology of pg crypts. In general, they were situated at the periphery of the epithelium and lacked access to the lumen, with consequent cystic enlargement and flattened epithelium. No obvious morphological changes were observed in the pg-derived areas of the uterine epithelium of this chimera. Our results provide evidence that pg cells can persist as proliferating stem cells in various tissues of early postnatal chimeras. They suggest that pg-derived stem cells may cease to proliferate in restricted areas of the gastrointestinal tract and in the uterine epithelium of pg <==> wt chimeras of advanced age.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Quimera/fisiologia , Partenogênese/fisiologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Sistema Digestório/citologia , Células Epidérmicas , Células Epiteliais , Epitélio/fisiologia , Feminino , Hibridização In Situ , Camundongos , Língua/citologia , Bexiga Urinária/citologia , Útero/citologiaRESUMO
Transgenic mice carrying the human cytomegalovirus immediate early gene promoter driving the E. coli lacZ gene displayed an unusual cell specific expression of beta-galactosidase during development. LacZ expression was first detected in cells lining the apex of the neural fold of day 8.5 embryos. By day 10 of gestation, expression was prominent in the spinal ganglia, the ganglia of cranial nerves V, VII, VIII, IX, and X, in a line of cells marking the ventrolateral pathway adjacent to the dermamyotome, and in a column of differentiated cells in the entire ventrolateral neural tube posterior to the mesencephalon. Expression was also found in the myotomes. Neural tube explants from day 8.5 embryos cultured in vitro showed lacZ expression in cells migrating away from the explant. We conclude that the HCMV-IEP-lacZ transgene is expressed in a subpopulation of neural crest cells and its early derivatives.
Assuntos
Citomegalovirus/genética , Regulação Viral da Expressão Gênica/genética , Genes Virais/genética , Regiões Promotoras Genéticas/genética , Animais , Sequência de Bases , DNA/genética , Embrião de Mamíferos/metabolismo , Escherichia coli/genética , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Crista Neural/citologia , Crista Neural/embriologia , Gravidez , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Mouse trophoblast giant cells undergo successive rounds of DNA replication resulting in amplification of the genome. It has been difficult to determine whether giant cell chromosomes are polyploid as in liver cells or polytene as in Dipteran salivary glands because the chromosomes do not condense. We have examined the pattern of hybridization of mouse giant cells with a variety of in situ chromosome markers to address this question. Hemizygous markers displayed one hybridization signal per nucleus in both diploid and giant cells, while homozygous markers displayed two signals per nucleus in both cell types. These patterns are consistent with cytological evidence indicating that giant cell chromosomes are polytene rather than polyploid. However, in contrast to the situation in Dipteran salivary glands, the two homologues do not appear to be closely associated. We conclude that the mechanism of giant cell DNA amplification involves multiple rounds of DNA replication in the absence of both karyokinesis and cytokinesis, and that sister chromatids, but not homologous chromosomes, remain closely associated during this process.
Assuntos
Cromossomos/embriologia , Trofoblastos/fisiologia , Animais , Blastocisto , Replicação do DNA , DNA Satélite , Marcadores Genéticos , Camundongos , Hibridização de Ácido NucleicoRESUMO
Transcription of the mouse hsp68 and hsc70 genes in embryonal carcinoma cells, various embryonic and extraembryonic tissues, and some adult tissues has been assessed using cloned probes to the mouse hsp68 gene. The results from Northern blots showed that both F9 and P19 cells respond in the expected manner to a heat shock. Hsp68 expression was only detected in heat-induced F9 and P19 cells. Hsc70 transcripts were present in uninduced cells and their levels increased after induction. In adult tissues, the hsp68 gene was expressed constitutively in the kidney. A different hsp68-like transcript was detected at significant levels in adult testes. Constitutive expression of hsp68 was observed in both the placenta (beginning at Day 8.5) and yolk sac (beginning at Day 11.5). No hsp68 expression was detected in embryonic tissues until Day 15.5. Expression of the mouse hsp68 gene during embryogenesis suggests that it may play some role in development.
Assuntos
Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Animais , Linhagem Celular , DNA/genética , Células-Tronco de Carcinoma Embrionário , Rim/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , Hibridização de Ácido Nucleico , RNA Mensageiro/metabolismo , Teratoma/metabolismo , Fatores de Tempo , Transcrição GênicaRESUMO
Using a Moloney leukemia virus vector containing the bacterial neo gene, we demonstrate that retrovirus vectors can be used to introduce genes into the mouse germ line. Infection of preimplantation embryos with the vector MLV-NEO.1 resulted in integration of neo sequences in approximately equal to 10% of the progeny mice. One of these animals, mouse F.2, contained approximately six MLV-NEO.1 proviruses at independent integration sites, each present at less than a single copy per cell. This mosaic mouse transmitted one of these proviruses to her offspring, producing a line of transgenic mice carrying a full-length, unrearranged MLV.NEO.1 provirus at a single chromosomal integration site. Mice homozygous at this MLV-NEO.1 locus have also been produced. No expression of the neo gene has been detected in the transgenic mice, either by screening of primary bone marrow or lung cells for resistance to G418 or by RNA transfer blot analysis of RNA from several tissues. In addition, the neo gene was found to be extensively methylated in the transgenic mice; however, treatment of primary cells with 5-azacytidine did not induce G418 resistance. The inactivity of the MLV-NEO.1 provirus in transgenic mice and potential means of eliciting neo expression under these conditions are discussed.
Assuntos
Genes Bacterianos , Engenharia Genética , Vetores Genéticos , Vírus da Leucemia Murina/genética , Camundongos/genética , Neomicina/farmacologia , Animais , Azacitidina/farmacologia , Resistência a Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes , Células Germinativas/fisiologia , Vírus Auxiliares/genética , Metilação , RNA Mensageiro/genéticaRESUMO
We have examined the effect of sodium butyrate on the levels of histone acetylation, the pattern of protein synthesis and the inducibility of heat shock polypeptides (hsps) in cultured trout fibroblasts. Maximal levels of histone acetylation are achieved upon treatment of these cells with 5 mM butyrate for 24 h. No significant changes in the pattern of protein synthesis, as detected by two-dimensional gel electrophoresis, are apparent under these conditions, although changes in the levels of three polypeptides are seen at shorter times of exposure to butyrate. Heat shock polypeptides are inducible at normal levels in butyrate-treated cells. This is in contrast to the ability of butyrate to inhibit the activation of steroid-inducible genes in some systems.
Assuntos
Proteínas de Choque Térmico/biossíntese , Histonas/fisiologia , Acetilação , Animais , Butiratos/farmacologia , Ácido Butírico , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , TrutaRESUMO
RTG-2 cells, a line of fibroblasts from rainbow trout (Salmo gairdnerii), are induced to synthesize a distinct set of heat-shock polypeptides after exposure to elevated temperature or to low concentrations of sodium arsenite. We isolated and characterized two cDNA sequences, THS70.7 and THS70.14, encoding partial information for two distinct species of 70-kilodalton heat shock polypeptide (hsp70) from these cells. These sequences are identical at 73.3% of the nucleotide positions in their regions of overlap, and their degree of sequence conservation at the polypeptide level is 88.1%. The two derived trout hsp70 polypeptide sequences show extensive homology with derived amino acid sequences for hsp70 polypeptides from Drosophila melanogaster and Saccharomyces cerevisiae. Northern blot analysis of RNA from arsenite-induced RTG-2 cells, with the trout hsp70 cDNAs as probes, revealed the presence of three hsp70 mRNA species. Southern blot analysis of trout testis DNA cleaved with various restriction endonucleases revealed a small number of bands hybridizing to the hsp70 cDNAs, suggesting the existence of a small family of hsp70 genes in this species. Finally, trout hsp70 cDNA sequences cross-hybridized with restriction fragments in genomic DNA from HeLa cells, bovine liver, Caenorhabditis elegans, and D. melanogaster.
Assuntos
DNA/análise , Proteínas de Choque Térmico/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Enzimas de Restrição do DNA , Drosophila melanogaster/genética , Fibroblastos/metabolismo , Peso Molecular , Hibridização de Ácido Nucleico , Saccharomyces cerevisiae/genética , Especificidade da Espécie , TrutaRESUMO
The heat-shock response has been characterized in cultured fibroblasts of the rainbow trout, Salmo gairdnerii. The response has been elicited by two different stress situations; cells were either subjected to higher temperatures than normal (27 to 29 degrees C as opposed to 22 degrees C) or were incubated in medium containing sodium arsenite (15 to 100 microM final concentration). The response of the cells to these conditions is to synthesize a set of new polypeptides, the "heat-shock polypeptides" (hsps), that are not present or present in extremely low amounts in noninduced cells. Furthermore, during prolonged arsenite induction, the synthesis of normal cellular proteins is repressed. In trout fibroblasts, at least six hsps are detectable. These range from 30 000 to 87 000 in molecular weight and are referred to as hsp30, hsp32, hsp42, hsp62, hsp70, and hsp87. The hsp30 and hsp70 components are the most abundant and can be visualized by Coomassie blue staining of gels after prolonged induction. The heat-shock response is a reversible process in trout cells. Results of in vitro translation of mRNA from induced cells indicate that the control of hsp induction may be at the transcriptional level. Hsp70 from trout comigrates with the major hsp from Drosophila melanogaster on sodium dodecyl sulfate - polyacrylamide gels, suggesting that this protein may be highly conserved in evolution.