RESUMO
Apoptosis pathways provide efficient safeguard mechanisms against cancer that are mediated via cell-intrinsic responses and immune-mediated extrinsic signals. Intrinsic pro-apoptotic pathways are largely controlled by p53 and Bcl-2 proteins, whereas the extrinsic induction of apoptosis is initiated by death ligands, such as tumour necrosis factor-alpha (TNF-alpha), CD95L/FasL and TNF-related apoptosis-inducing ligand (TRAIL), or by granzyme B. Initiation of these pathways results in the induction of a caspase cascade leading to cell death. The inactivation of pro-apoptotic pathways is elementary for tumourigenesis and may be responsible for therapy resistance. Thus, apoptosis-based strategies represent important tools for the development of effective tumour therapies. The aim of these therapies is to restore p53 activity, downregulate anti-apoptotic Bcl-2 proteins or NF-kappaB activity, and to upregulate extrinsic, death receptor-mediated pathways. The initial results of apoptosis-based strategies are proving promising. Also, topical treatments for actinic keratosis (AK), such as cyclo-oxygenase-2 inhibitors (e.g. diclofenac 3% gel), have been shown to trigger pro-apoptotic pathways. There is hope that pro-apoptotic strategies will lead to pronounced therapeutic success against skin cancer. Importantly, the involvement of the different pro-apoptotic pathways in specific tumour types needs to be unravelled and understood in order to evaluate drug effectiveness, as well as to modify and optimise therapeutic approaches.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Comunicação Celular , Humanos , Neoplasias Cutâneas/imunologiaRESUMO
In addition to having anti-inflammatory activities, nonsteroidal anti-inflammatory drugs (NSAIDs) also inhibit neoplastic cell proliferation by inducing apoptosis. Diclofenac is the anti-neoplastic compound in diclofenac 3% gel (Solaraze) used for topical treatment of actinic keratosis (AK). Main target of NSAIDs seems to be the inhibition of cyclo-oxygenase-2 (COX-2), which is overexpressed in several epithelial tumours and catalyses the synthesis of prostaglandins. The precise mechanism of action of diclofenac in cutaneous cells is still unclear, but induction of apoptosis is a key effect of anti-neoplastic drugs, including NSAIDs. In this paper we give an overview of the anti-tumoural activities of NSAIDs with emphasis on induction of apoptosis. Cyclo-oxygenase-2-mediated synthesis of prostaglandin E(2) (PGE(2)) leads to activation of mitogen-activated protein kinase (MAPK), as well as phosphatidylinositol 3-kinase (PI3K)/Akt pathways. Induction of the anti-apoptotic Bcl-2 and Mcl-1, as well as activation of the caspase-8 inhibitor cFLIP have been reported. In addition, altered lipid concentrations in the cytoplasmic membrane may modulate death receptor activities. Downregulation of both the intrinsic mitochondrial and the extrinsic pathways have been reported. Our data demonstrate induced apoptosis and activation of the caspase cascade in three of four cutaneous squamous cell carcinoma (SCC) cell lines, after treatment with diclofenac plus hyaluronic acid and diclofenac alone; one cell line remained nonresponsive. The effects were less pronounced in normal keratinocytes and cytotoxic effects were not seen. Detailed analysis of apoptosis pathways employed by diclofenac in these cells may help to improve therapeutic strategies and to overcome possible mechanisms that are involved in nonresponsiveness.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Apoptose/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Ácido Hialurônico/uso terapêutico , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Adjuvantes Imunológicos/uso terapêutico , Indutores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Diclofenaco/uso terapêutico , Humanos , Ceratose/tratamento farmacológico , Prostaglandinas/biossínteseRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted considerable attention as a novel anticancer agent. However, its efficiency may be diminished by occurring resistance in cancer cells. The mechanisms of TRAIL resistance in melanoma are still unsolved. Here we show for the first time that TRAIL-induced activation of NF-kappaB occurs in apoptosis-sensitive melanoma cell lines through TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4), whereas TRAIL failed to activate nuclear factor kappa B (NF-kappaB) in melanoma cells positive only for TRAIL receptor 2/death receptor 5 (TRAIL-R2/DR5). However, activation of NF-kappaB by TRAIL was not associated with enhanced expression of antiapoptotic factors: cellular FLICE-inhibitory protein (c-FLIP), Bcl-x(L), X-linked inhibitor of apoptosis protein (XIAP), Survivin, Livin. Rather in one of the cell lines, TRAIL induced the downregulation of DR4. In an established cell culture model for TRAIL resistance and regained TRAIL sensitivity, resistance was neither associated with increased NF-kappaB activity by TRAIL nor by an increased expression of antiapoptotic proteins. However, significant downregulation of caspase-8, caspase-10 and of DR4 was characteristic for TRAIL-resistant, DR4-positive melanoma cells, and regained TRAIL sensitivity coincided with re-expression of these factors. Sensitivity was also largely retained after their exogenous overexpression. Thus, initiator caspases and DR4 rather than NF-kappaB may control melanoma cell sensitivity to TRAIL, and strategies, which result in their upregulation, may be useful for enhancement of TRAIL sensitivity.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Caspases Iniciadoras/metabolismo , Regulação para Baixo , Melanoma/metabolismo , NF-kappa B/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Caspases Iniciadoras/genética , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Humanos , RNA Interferente Pequeno/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Sensibilidade e Especificidade , Regulação para Cima/efeitos dos fármacosRESUMO
Pro- and antiapoptotic proteins of the large Bcl-2 family are critical regulators of apoptosis via the mitochondrial pathway. Whereas antiapoptotic proteins of the family share all four Bcl-2 homology domains (BH1-BH4), proapoptotic members may lack some of these domains, but all so far described proapoptotic Bcl-2 proteins enclose BH3. The bcl-x gene gives rise to several alternative splice products resulting in proteins with distinct functions as the antiapoptotic Bcl-xL and proapoptotic Bcl-xS. Here, we describe a novel Bcl-x splice product of 138 amino acids termed Bcl-xAK (Atypical Killer), which encloses the Bcl-2 homology domains BH2 and BH4 as well as the transmembrane domain, but lacks BH1 and BH3. Weak endogenous expression of Bcl-xAK was seen in melanoma and other tumor cells. Interestingly, its overexpression by applying a tetracycline-inducible expression system resulted in significant induction of apoptosis in melanoma cells, which occurred in synergism with drug-induced apoptosis. After exogenous overexpression, Bcl-xAK was localized both in mitochondrial and in cytosolic cell fractions. By these findings, a completely new class of Bcl-2-related proteins is introduced, which promotes apoptosis independently from the BH3 domain and implies additional, new mechanisms for apoptosis regulation in melanoma cells.
Assuntos
Processamento Alternativo , Apoptose , Melanoma/patologia , Neoplasias Cutâneas/patologia , Proteína bcl-X/fisiologia , Sequência de Aminoácidos , Antibacterianos/farmacologia , Clonagem Molecular , Citosol/metabolismo , Doxiciclina/farmacologia , Células HeLa , Humanos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Frações SubcelularesRESUMO
Control of translation initiation was recognised as a critical checkpoint for cell proliferation and tumorigenesis. In human melanoma cells, we have previously reported consistent overexpression of translation initiation factor eIF-4A1. Here, we investigated by transfection of antisense constructs its significance for the control of melanoma cell growth. The tetracycline-inducible expression system was established in melanoma cells, and three fragments of the 5'-, central-, and 3'-portion of the eIF-4A1 cDNA were subcloned in antisense and in sense orientation after a tetracycline inducible promoter. Significant proliferation decrease was obtained after transient transfection and induction of antisense RNA directed against the 5'- and the central portion (up to 10%), whereas, no effects were seen after induction of the 3'-fragment and the sense controls. Cell clones stably transfected with the central antisense fragment revealed after doxycycline induction reduced expression of endogeneous eIF-4A1 mRNA correlated with decreased proliferation rates (up to 6%). These data demonstrate the applicability of antisense strategies against translation factors in melanoma cells. Translation initiation factor eIF-4A1 contributes to the control of melanoma cell proliferation and may be taken into consideration when scheduling new therapeutic approaches targeting the translational control.
Assuntos
Melanoma/patologia , Fatores de Iniciação de Peptídeos/genética , RNA Antissenso/fisiologia , Neoplasias Cutâneas/patologia , Antibacterianos/farmacologia , Divisão Celular , Regulação para Baixo , Fator de Iniciação 4A em Eucariotos , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/metabolismo , Polirribossomos/metabolismo , RNA Mensageiro/metabolismo , Neoplasias Cutâneas/metabolismo , Tetraciclinas , Transfecção , Células Tumorais CultivadasRESUMO
BACKGROUND: In embryonic kidneys, mesenchymal cells convert into epithelium in response to an induction by the tip of the ureter bud. Metanephric mesenchyme can also be induced to convert into epithelium in vitro. It is a model system to identify genes that could be important for epithelial development. METHODS: By differential screening of a cDNA library made from mesenchymes induced in transfilter cultures by embryonic spinal cord for 24 hours, we selected cDNA clones representing genes that were preferentially expressed in 24-hour-induced mesenchyme and not in uninduced mesenchyme. The sequence of one clone was determined and used to obtain the sequence of a complete open reading frame. By Northern blotting and in situ hybridization, the expression of the mRNA in embryonic kidneys was determined. RESULTS: We report the sequence and expression pattern of a marker for the 24-hour-induced state, mouse nuclear pore membrane glycoprotein 210 (mPOM210). The deduced 1886 amino acid sequence shows a 95% identity to the sequence of rat gp210. Northern blotting revealed a single 7.5 kb mRNA in 24-hour-induced mesenchyme, whereas message levels were fourfold to fivefold lower in uninduced mesenchyme. In situ hybridization of in vivo development confirmed the preferential expression of mPOM210 in epithelial cells. In the kidney, expression was seen in both the epithelium derived from the ureteric tree and the mesenchyme-derived epithelium. In other tissues of 13-day-old embryos, expression was also confined to the epithelium. In nervous tissues, the olfactory epithelium and walls of the lateral ventricle were the most prominently stained. Weak expression was seen in the heart. CONCLUSIONS: mPOM210 mRNA is an early marker for developing epithelial cells. Furthermore, our results suggest that nuclear pore membrane proteins could be more cell-type specific than previously anticipated.
Assuntos
DNA Complementar/genética , Rim/embriologia , Rim/metabolismo , Glicoproteínas de Membrana/genética , Proteínas Nucleares/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Biomarcadores , Polaridade Celular , Clonagem Molecular , Primers do DNA/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Complexo de Proteínas Formadoras de Poros Nucleares , Gravidez , Ratos , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Transgenic plants of Nicotiana benthamiana expressing single chain antibody fragments (scFv) specific for the coat protein of beet necrotic yellow vein virus (BNYVV) and non-expressing control plants were inoculated with BNYVV mechanically and by means of the vector Polymyxa betae. The scFv were presumably expressed in the endoplasmic reticulum (ER). The average time needed for infections to become detectable was longer in the scFv-expressing plants than in the non-expressing control plants. In addition, the scFv-expressing plants were partially protected against the pathogenic effects exerted by the virus on N. benthamiana plants in the late stages of infection.
Assuntos
Capsídeo/imunologia , Fragmentos de Imunoglobulinas/biossíntese , Nicotiana/virologia , Doenças das Plantas/virologia , Vírus de Plantas/imunologia , Plantas Tóxicas , Vírus de RNA/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Citoplasma/imunologia , Retículo Endoplasmático/imunologia , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/imunologia , Vírus de Plantas/fisiologia , Plantas Geneticamente Modificadas , Vírus de RNA/fisiologia , Proteínas Recombinantes/biossíntese , Nicotiana/imunologiaRESUMO
Laminins, found predominantly in basement membranes, are large glycoproteins consisting of different subsets of alpha, beta and gamma chain subunits. To resolve conflicting data in the literature concerning coexpression of alpha 1 and beta 2 chains, expression of alpha 1 chain was studied with two different antisera against the E3 fragment of laminin alpha 1 chain. Expression of the alpha 1 chain was seen in several types of epithelial basement membranes throughout development, but its expression in rat glomerular basement membranes and some other types of epithelial basement membranes occurred only during early stages of development. By contrast, beta 2 chains were detected by immunofluorescence only during advanced stages of glomerulogenesis and vascular development. By Northern and Western blots, beta 2 chains were detected somewhat earlier, but in situ hybridization revealed that beta 2 chain was also confined to vasculature during the earlier stages. It thus seems that, in the tissues studied here, the expression of alpha 1 and beta 2 chains was mutually exclusive. To explore whether the newly described alpha 5 chain is expressed in locations lacking alpha 1 chain, expression of alpha 5 chain was studied by Northern blots and in situ hybridization. The alpha 5 chain was not uniformly expressed in all embryonic epithelial cell types but was present mainly in epithelial sheets which produce very little alpha 1 chain. There also appeared to be a developmental trend, with alpha 1 chain appearing early and alpha 5 later, in maturing epithelial sheets. The alpha 5 chain could be a major alpha chain of the adult glomerular basement membrane.
Assuntos
Envelhecimento , Vasos Sanguíneos/metabolismo , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento , Rim/irrigação sanguínea , Rim/metabolismo , Laminina/biossíntese , Miocárdio/metabolismo , Animais , Membrana Basal/fisiologia , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/crescimento & desenvolvimento , Epitélio/embriologia , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Coração/embriologia , Coração/crescimento & desenvolvimento , Hibridização In Situ , Rim/embriologia , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Camundongos , RatosRESUMO
The coding sequences for the variable regions of heavy and light chains of monoclonal antibodies (mAbs) to beet necrotic yellow vein virus (BNYVV) coat protein (cp) or the 25 kDa nonstructural protein (P25) were cloned into the pCOCK vector and expressed as single-chain antibody fragments (scFv) in Escherichia coli. For expression in higher plants the scFv were targeted either to the secretory pathway by including the sequences encoding the pectate lyase B (PelB) or the phytohemagglutinin (PHA) signal peptides in the vector constructs or they were targeted to the cytoplasm by omitting a signal peptide-encoding sequence from the constructs. The scFv were detected mainly in plants in which the PHA signal peptide had been used for targeting demonstrating for the first time the usefulness of this peptide for enabling scFv expression in plants. The scFv were not secreted into the culture fluids of suspension cultures, but were retained in the cells. The amount of expression of scFv in the best expressing plants was at least as high as in bacterial culture supernatants. In a dot blot immunoassay, 0.4 ng BNYVV cp or 0.8 ng P25 were detected by the respective scFv either from E. coli or from plants. The majority of the 21 plants expressing cp-specific scFv had near-normal growth whereas the three plants expressing P25-specific scFv grew poorly and did not form roots.
Assuntos
Anticorpos Antivirais/imunologia , Capsídeo/imunologia , Fragmentos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/imunologia , Vírus de Plantas/imunologia , Vírus de RNA/imunologia , Anticorpos Antivirais/genética , Especificidade de Anticorpos , Capsídeo/metabolismo , Clonagem Molecular , Escherichia coli , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Dados de Sequência Molecular , Plantas Tóxicas , Sinais Direcionadores de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Nicotiana , Proteínas não Estruturais Virais/genéticaRESUMO
The gene of a bacterial lysine decarboxylase (ldc) fused to a rbcS transit peptide coding sequence (tp), and under the control of the CaMV 35S promoter, was expressed in hairy root cultures of Nicotiana tabacum. The fusion of the ldc to the targeting signal sequence improved the performance of the bacterial gene in the plant cells in many respects. Nearly all transgenic hairy root cultures harbouring the 35S-tp-ldc gene contained distinctly higher lysine decarboxylase activity (from 1.5 to 30 pkat LDC per mg protein) than those which had been transformed with constructs in which the gene had been directly cloned behind the CaMV 35S promoter. The higher enzyme activity led to the accumulation of up to 0.7% cadaverine on a dry mass basis. In addition, part of the cadaverine pool was used for increased biosynthesis of anabasine, an alkaloid which was hardly detectable in control cultures. The best line contained anabasine levels of 0.5% dry mass, which could be further be enhanced by feeding of lysine.
Assuntos
Proteínas de Bactérias/genética , Carboxiliases/genética , Nicotiana/citologia , Raízes de Plantas/metabolismo , Plantas Tóxicas , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Anabasina/metabolismo , Proteínas de Bactérias/metabolismo , Cadaverina/metabolismo , Carboxiliases/metabolismo , Resistência a Canamicina/genética , Cinética , Vírus do Mosaico/genética , Técnicas de Cultura de Órgãos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas/metabolismoRESUMO
Recent biochemical studies suggested that the extracellular matrix protein nidogen is a binding molecule linking together basement membrane components. We studied its expression and role during development. By immunofluorescence and northern blotting, nidogen was found early during epithelial cell development of kidney and lung. Yet, in situ hybridization revealed that nidogen was not produced by epithelium but by the adjacent mesenchyme in both organs. Binding of mesenchymal nidogen to epithelial laminin may thus be a key event during epithelial development. This is supported by antibody perturbation experiments. Antibodies against the nidogen binding site on laminin B2 chain perturbed epithelial development in vitro in embryonic kidney and lung. Mesenchymal nidogen could be important for early stages of epithelial morphogenesis.
Assuntos
Epitélio/fisiologia , Glicoproteínas de Membrana/fisiologia , Mesoderma/fisiologia , Animais , Sequência de Bases , Membrana Basal/fisiologia , Northern Blotting , Técnicas de Cultura , Epitélio/química , Imunofluorescência , Hibridização In Situ , Rim/química , Rim/embriologia , Laminina/fisiologia , Pulmão/química , Pulmão/embriologia , Glicoproteínas de Membrana/análise , Camundongos , Dados de Sequência Molecular , Morfogênese/fisiologia , Sondas de OligonucleotídeosRESUMO
Several hairy root cultures of Nicotiana tabacum varieties, carrying two direct repeats of a bacterial lysine decarboxylase (ldc) gene controlled by the cauliflower mosaic virus (CaMV) 35S promoter expressed LDC activity up to 1 pkat/mg protein. Such activity was, for example, sufficient to increase cadaverine levels of the best line SR3/1-K1,2 from ca. 50 micrograms (control cultures) to about 700 micrograms/g dry mass. Some of the overproduced cadaverine of this line was used for the formation of anabasine, as shown by a 3-fold increase of this alkaloid. In transgenic lines with lower LDC activity the changes of cadaverine and anabasine levels were correspondingly lower and sometimes hardly distinguishable from controls. Feeding of lysine to root cultures, even to those with low LDC activity, greatly enhanced cadaverine and anabasine levels, while the amino acid had no or very little effect on controls and LDC-negative lines.
Assuntos
Anabasina/metabolismo , Cadaverina/biossíntese , Carboxiliases/genética , Nicotiana/genética , Plantas Tóxicas , Northern Blotting , Southern Blotting , Carboxiliases/biossíntese , Clonagem Molecular , Técnicas de Cultura , Escherichia coli , Vetores Genéticos , Plantas Geneticamente Modificadas , Mapeamento por Restrição , Rhizobium/enzimologia , Nicotiana/metabolismo , Nicotiana/microbiologiaRESUMO
Laminin is a large glycoprotein of basement membranes. The best described laminin from a mouse tumor contains three polypeptide chains (A, B1 and B2), but there is recent evidence that some cell types produce laminin isoforms lacking the A chain. We have here studied the occurrence of the isoforms during mouse organogenesis. In all tissues studied, the A chain mRNA and polypeptide were more weakly expressed than those of the B chains. Laminin A chain polypeptides showed a much more restricted tissue distribution than the B chains. Laminin A chain polypeptide was mainly detected in basement membranes of epithelial cells, suggesting that this chain is important for morphogenesis of epithelial sheets. Most endothelial basement membranes and all embryonic mesenchyme matrices studied seemed to lack the A chain even though they contained B chains. Several of the cells producing laminin devoid of A chain seem to produce other polypeptides that become complexed to the B chains. With an anti-laminin antiserum, which in immunoblots reacts only with A and B polypeptide chains, additional polypeptides of 160 and 190 x 10(3) Mr were co-precipitated from all tissues studied. In developing heart, a polypeptide of 300 x 10(3) Mr was co-precipitated in addition. Our data suggest that these laminin-associated polypeptides are not formed by a differential splicing of the known A chain mRNA. Northern blotting of poly (A)+ RNA showed only 10kb A chain transcripts but no truncated forms. We conclude that several cell types in the mouse embryo produce laminin variants that lack the 400 x 10(3) Mr A chain. Since a major cell binding site of laminin contains parts of the A chain, the variants should differ in biological function from laminin containing this A chain.
Assuntos
Embrião de Mamíferos/fisiologia , Laminina/fisiologia , Animais , Olho/embriologia , Imunofluorescência , Coração/embriologia , Immunoblotting , Intestinos/embriologia , Laminina/análise , Fígado/embriologia , Pulmão/embriologia , Camundongos , Morfogênese/fisiologia , Medula Espinal/embriologia , Cauda/embriologia , Timo/embriologiaRESUMO
Replication-dependent histone mRNAs are prime examples of nonpolyadenylated mRNAs. We isolated and characterized cDNAs and a genomic clone for a replication-dependent histone H2A.1 mRNA which segregated into the poly(A)+ fraction during mRNA isolation through an oligo(dT)-cellulose column. However, the results of sequencing of the genomic clone suggested that the mRNA did not contain a poly(A) tail. Instead, the genomic sequence revealed a nonterminal oligo(A) tract directly upstream from the typical 3'-terminal hairpin loop of replication-dependent histone mRNAs. The nonterminal oligo(A) tract consisted of 14 adenylate residues interrupted by one guanylate residue (A4GA10). We concluded that this short oligo(A) stretch mediated binding of the mRNA to oligo(dT) even after stringent washes with 0.1 M NaCl, indicating that rather short oligo(A) sequences can ensure binding to oligo(dT)-cellulose. The cDNA and genomic clones contained an AAATAAG sequence at the end of the coding region. It has been suggested that this sequence contains a polyadenylation signal in some yeast and mouse transcripts, but it does not function as a polyadenylation signal in the histone transcript described in this paper.
Assuntos
Genes , Histonas/genética , Poli A/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Divisão Celular , Células Cultivadas , Clonagem Molecular , DNA/genética , Embrião de Mamíferos , Biblioteca Gênica , Rim/metabolismo , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Técnicas de Cultura de Órgãos , Baço/citologiaRESUMO
Three polypeptide chains, A, B1, and B2, have been described for mouse laminin, a basement membrane protein. We studied expression of laminin A, B1, and B2 mRNA in the developing mouse kidney. Induction of kidney mesenchyme differentiation in vitro led to an increased expression of B1 and B2 chain mRNA on day 1 of development. In contrast, expression of A chain mRNA increased on day 2, when epithelial cell polarization begins. Laminin A mRNA and polypeptide were expressed only by epithelia during in vivo development as well. Some polarized cell types producing basement membrane (endothelium, some adult epithelia) lacked the A chain mRNA and polypeptide, although they did express B chains. Laminin with the 400 kd A chain is therefore a transient form appearing at specific sites of kidney morphogenesis, whereas isoforms with a different A chain or without it have a more widespread distribution.
Assuntos
Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica , Genes , Rim/embriologia , Laminina/genética , RNA Mensageiro/genética , Transcrição Gênica , Animais , Anticorpos Monoclonais , Northern Blotting , Embrião de Mamíferos , Epitélio/metabolismo , Imunofluorescência , Rim/metabolismo , Laminina/biossíntese , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos , Hibridização de Ácido Nucleico , Técnicas de Cultura de ÓrgãosRESUMO
Each of the four hydroxamate compounds, ferrichrome, aerobactin, rhodotorulic acid, and coprogen, known to transport ferric iron into Escherichia coli requires a specific outer membrane receptor protein. In addition, common transport functions for all four ferric hydroxamate compounds have been identified in the 3.5-min region of the linkage map and designated fhu. The fhu region was cloned into pBR322. By restriction analysis, Tn5 insertion mutations, and complementation studies between plasmid fragments and chromosomal mutants at least four loci in the order fhuA fhuC fhuD fhuB were found. The genetic products were determined in maxicells and minicells. fhuA codes for the known 78,000-dalton receptor protein and the 81,000-dalton precursor in the outer membrane, fhuC codes for a 30,000-dalton protein, and fhuD encodes a 26,000-dalton protein in the cytoplasmic membrane. No protein(s) could be assigned to the fhuB region. Truncated proteins derived from partial fhuA genes (68,000, 42,000, and 39,000 daltons) and a partial fhuD gene (24,000 daltons) and the strong polar effect on the expression of the genes indicated the direction of transcription to be from fhuA to fhuD.