RESUMO
The six closely related and clustered rat gamma-crystallin genes, the gammaA- to gammaF-crystallin genes, are simultaneously activated in the embryonic lens but differentially shut down during postnatal development with the gammaB-crystallin gene, the last one to be active. We show here that developmental silencing of the gammaD-crystallin promoter correlates with delayed demethylation during lens fiber cell differentiation. Methylation silencing of the gammaD-crystallin promoter is a general effect and does not require the methylation of a specific CpG, nor does methylation interfere with factor binding to the proximal activator. In later development, the gammaD-crystallin promoter is also shut down earlier by a repressor that footprints to the -91/-78 region. A factor with identical properties is present in brain. Hence, a ubiquitous factor has been recruited as a developmental regulator by the lens. All gamma-crystallin promoters tested contain upstream silencers, but at least the gammaB-crystallin silencer is distinct from the gammaD-crystallin silencer. The gamma-crystallin promoters were found to share a proximal activator (the gamma-box; around -50), which behaves as a MARE. The gammaB-box is recognized with much lower avidity than the gammaD-box. By swapping elements between the gammaB- and the gammaD-crystallin promoter, we show that activation by the gammaB-box requires a directly adjacent -46/-38 AP-1 consensus site. These experiments also uncovered another positive element in the gammaD-crystallin promoter, around -10. In the context of the gammaD-crystallin promoter, this element is redundant; in the context of the gammaB-crystallin promoter, it can replace the -46/-38 element.
Assuntos
Cristalinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas , Animais , Animais Recém-Nascidos , Sequência de Bases , Sítios de Ligação , Cristalinas/metabolismo , DNA , Dados de Sequência Molecular , Mutagênese , Ratos , Ratos Wistar , Sequências Reguladoras de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Fator de Transcrição AP-1/metabolismoRESUMO
Fibroblast growth factors (FGFs) are the only known factors that can induce differentiation of the mammalian lens epithelial cell, while insulin acts only as a mitogen, not as a morphogen. We show here that insulin enhances expression of the alphaA-crystallin gene in lens epithelial cells and induces the synthesis of lens fibre cell specific betaB2- and gamma-crystallins in early differentiated fibre cells. Different signal transduction pathways are required for bFGF or insulin maintained fibre cell differentiation. A 15 min preincubation with bFGF was sufficient for the lens epithelial cells to become competent to undergo insulin maintained differentiation. The phorbol ester TPA could replace bFGF. The bFGF instructed competence to differentiate decays with a half-life of about 30 h. Hence, bFGF and insulin can act in concert to produce a differentiated phenotype even when they are not present simultaneously.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Insulina/metabolismo , Cristalino/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cristalino/citologia , Fenótipo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Mutations, expected to affect the intracellular routing, i.e. additional nuclear localization sequences (NLS; the natural 23 kDa isoform and a 17D27R mutant) and/or a deletion of amino acids 26-29 (23 delta 26-29 and 17 delta 26-29), were introduced in basic fibroblast growth factor (bFGF). The mutants were assayed for their mitotic activity and their capacity to induce a tissue-specific response in human umbilical vein endothelial cells [HUVECs; induction of urokinase plasminogen activator receptor (u-PAR)], or in rat lens epithelial cells (fibre cell differentiation). In HUVECs, the 17D27R mutant had wild type activity, the 23 kDa and the delta 26-29 proteins were impaired in the induction of both mitosis and u-PAR. The delta 26-29 proteins, but not the 23 kDa protein or 17D27R mutant, were also impaired in receptor binding in that they bound only to a subset of receptors. The concentration of 17 kDa bFGF required for half maximal u-PAR response was 30 fold higher than for the half maximal 3H-thymidine incorporation. Addition of an NLS to bFGF strongly inhibited the induction of fibre cell differentiation, though it had little effect on the stimulation of DNA synthesis. The 17 delta 26-29 kDa mutant had wild type differentiation activity but was a poor mitogen for lens epithelial cells.
Assuntos
Células Epiteliais/citologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Mutação/fisiologia , Receptores de Superfície Celular/biossíntese , Animais , Células COS , Diferenciação Celular , Células Cultivadas , Cristalinas/biossíntese , DNA/biossíntese , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Células Epiteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Cristalino/citologia , Cristalino/metabolismo , Mitógenos/fisiologia , Sinais de Localização Nuclear/fisiologia , Ratos , Ratos Wistar , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes de Fusão , Deleção de Sequência , Veias UmbilicaisRESUMO
In duck, the housekeeping enzyme lactate dehydrogenase B (LDH-B) and the lens structural protein epsilon-crystallin are encoded by the same single copy gene. Transcription of the gene is initiated from two closely spaced start sites, at -28 and +1. The usage of the downstream site is greatly enhanced in lens. Deletion mapping of the promoter shows that the region -70/+18 specifies the enhanced promoter activity in the lens. A critical role is played by the consensus Sp1 binding site at -50; mutation of this site abolishes lens-preferred expression. Deletion of the +1 transcription initiation site also leads to a decrease in lens-preferred expression, which can be restored by moving the -28 transcription initiation site downstream. By band shift experiments, supershift mobility assays and methylation interference assays, Sp1 was shown to bind to the Sp1 consensus binding site at -50 using either heart or lens nuclear extracts. Co-expression of Sp1 or Sp1-like factors inhibited the activity of an LDH-B/epsilon-crystallin promoter construct by approximately 60% in lens and by 40% in heart cells. Co-expression of Pax-6, a transcription factor shown to be involved in the lens-enhanced expression of a number of other crystallin genes, did not influence the promoter activity of the -130/+650 LDH-B/epsilon-crystallin promoter construct. In contrast to other crystallin promoters, the LDH-B/epsilon-crystallin promoter does not appear to contain a lens-specific element, rather our data lead to a model in which a factor transmitting the effect of Sp1, bound at -50, to the transcription initiation complex is responsible for the lens-preferred expression of the LDH-B/epsilon-crystallin promoter.
Assuntos
Cristalinas/genética , Proteínas de Ligação a DNA/fisiologia , Patos/genética , Proteínas de Homeodomínio , L-Lactato Desidrogenase/genética , Cristalino/fisiologia , Fator de Transcrição Sp1/fisiologia , Animais , Células Cultivadas , Pegada de DNA , Proteínas do Olho , Regulação da Expressão Gênica no Desenvolvimento , Isoenzimas , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras , Transcrição GênicaRESUMO
The rat beta B2-crystallin gene is active only during a specific stage of the differentiation of rat lens fibre cells directed by basic fibroblast growth factor. The regulatory elements that determine the transient activity of this gene are located in the -750/-123 region and in the first intron. Singly, these elements act as silencers, together they constitute an enhancer that is active only during the specific differentiation stage. An additional silencer is found between -123 and -77. The proximal promoter region contains a Pax-6 binding site at -65/-51. In vitro, binding to this site could be detected but, according to in vivo footprinting experiments, this site is not occupied in the endogenous gene. Furthermore, co-expression of Pax-6 did not enhance promoter activity. Finally, mutation or deletion of this site did not affect promoter activity: the region -37/+10 sufficed for basal promoter activity. The cooperation between the -750/ -123 region and the first intron of the beta B2-crystallin gene not only determines the differentiation stage-specific activity of the gene, but also contributes to the highly increased expression in lens cells compared with non-lens cells.
Assuntos
Cristalinas/genética , Elementos Facilitadores Genéticos , Cristalino/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular/genética , Células Cultivadas , Sequência Consenso , DNA , Cristalino/citologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ratos , Sequências Reguladoras de Ácido Nucleico , Homologia de Sequência de Aminoácidos , Transcrição Gênica/genéticaRESUMO
The transcriptional activation of tissue-specific genes during terminal differentiation must be preceded by the priming of the chromatin and the appearance of the required transacting factors. We have timed these events for the transcriptional activation of the rat gamma D-crystallin gene, a lens fiber cell-specific gene that encodes a structural lens protein, during the (basic fibroblast growth factor (bFGF)-induced) in vitro differentiation of rat lens fiber cells. In vitro, in the presence of bFGF only, the endogenous gamma D mRNA accumulates between Day 10 and Day 15. When insulin is added as well, the differentiation process is accelerated and gamma D mRNA starts to accumulate at Day 8. Demethylation of the gamma D promoter region, as assessed by measuring the methylation state of the ThaI site at -16, occurs much sooner, within 1 day. By genomic footprinting, the first protein interaction with the promoter region was visible at Day 8; full occupancy of the promoter region could be detected only at Day 12. The genomic footprint identified four putative regulatory regions: -141/-131, -88/-71, -55/-45, and -15/-4. Site-directed mutagenesis of the G residues at -55 and -46 resulted in a three- to fivefold decrease in promoter activity of transfected gamma D/CAT reporter genes and also abolished interaction with nuclear extract factor(s). A G-->T mutation at -43 had no effect. The -55/-45 footprint thus derives from a proximal activator. The -88/-71 footprint identifies a silencer of the gamma D promoter in late fiber cell differentiation, as a tetramer of the -85/-67 sequence silenced a tk/CAT construct when transfected into fiber cells at a late stage, but not at an early stage, of in vitro differentiation. To time the appearance of regulatory factors, the activity of a -73/+45 gamma D/CAT (containing the activator region) and of a -1100/+45 gamma D/CAT construct was measured during fiber cell differentiation. The -73/+45 construct was active between Day 5 and Day 14, with a maximum at Day 12. The additional sequence information present in the -1100/+45 construct constrained gamma D promoter activity to between Day 8 and Day 13, with a maximum at Day 10. We conclude that the phased appearance of transacting factors during lens fiber cell differentiation controls the timing of first the activation and then the shutdown of the gamma D-crystallin gene promoter.
Assuntos
Cristalinas/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Cristalino/crescimento & desenvolvimento , Animais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Cristalinas/genética , DNA/química , Pegada de DNA , Análise Mutacional de DNA , Sinergismo Farmacológico , Fator 2 de Crescimento de Fibroblastos/farmacologia , Insulina/farmacologia , Cristalino/citologia , Cristalino/efeitos dos fármacos , Metilação , Modelos Genéticos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Fatores de Tempo , Transativadores/biossíntese , Ativação TranscricionalAssuntos
Glicoproteínas de Membrana/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento de Epitopos , Variação Genética , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Plasmodium falciparum/imunologia , Polimorfismo Conformacional de Fita Simples , Proteínas de Protozoários/imunologia , Vacinas ProtozoáriasRESUMO
We studied the expression of the angiogenic factor vascular permeability factor) (VPF, also called vascular endothelial growth factor), in human melanoma cells in vitro and in vivo. Melanoma lines that develop tumors with a low metastatic potential in nude mice were found to have low expression levels of VPF in vitro, and the VPF expression levels in melanoma lines that yield highly metastatic xenografts were high. However, in vivo the correlation between VPF mRNA levels and the frequency of metastasis was lost; in all xenografts equally high levels of VPF mRNA were found, independent of the parental cell line. Hence, in vivo VPF gene expression was upregulated in the low expressing lines. The external factor responsible for this induction may be hypoxia, given that we found that low oxygen tension caused a (reversible) increase in the VPF mRNA levels in otherwise low expressing melanoma lines in vitro. A melanoma line with an inducible VPF expression was engineered into a line with a constitutive VPF expression. In the xenografts from this line a change in the vascular architecture was seen, indicating that the pattern or the level of VPF expression is important for tumor angiogenesis in melanoma xenografts.
Assuntos
Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/fisiologia , Linfocinas/biossíntese , Linfocinas/fisiologia , Melanoma/irrigação sanguínea , Melanoma/metabolismo , Neovascularização Patológica/metabolismo , Processamento Alternativo , Animais , Sequência de Bases , Northern Blotting , Humanos , Melanoma/secundário , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Transplante de Neoplasias/fisiologia , Especificidade da Espécie , Transfecção , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Vascular permeability factor, or vascular endothelial growth factor (VPF/VEGF) is an important factor in the regulation of vascular growth and vascular permeability. VPF is a secreted, dimeric protein and has 8 cysteine residues conserved with platelet-derived growth factor (PDGF). To study the role of some of these cysteine residues in maintaining the structure and function of VPF, we replaced the codons for the second, third, fourth, and fifth cysteine by serine codons, and expressed the mutant proteins in a mammalian expression system. Cysteine residues 2 and 4 in VPF were found to be directly involved in anti-parallel interchain disulfide bonds, as in PDGF. VPF mutants lacking one of these cysteins were severely impaired in their S-linked dimerization, while upon coexpression of both mutants the ability to form dimers was restored. The VPF mutants lacking cysteine residue 2 or 4 also competed poorly for receptor binding of labeled VPF and had low biological activity, but these defects were also complemented by coexpressing the two mutants, indicating that for efficient receptor binding and activation VPF needs to be a covalent dimer, unlike PDGF-BB. Furthermore, cysteine residue 5 was found to be essential for VPF dimerization and activity, while the mutant lacking cysteine residue 3 was only mildly affected in its ability to dimerize and had partial biological activity.
Assuntos
Cisteína/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Serina/metabolismo , Ligação Competitiva , Biopolímeros , Fatores de Crescimento Endotelial/química , Fatores de Crescimento Endotelial/genética , Linfocinas/química , Linfocinas/genética , Mutação , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The modular construction of the eye lens beta gamma-crystallins makes them good candidates for protein engineering to ascertain the rules of assembly of oligomers. X-ray studies have shown that although the polypeptide chains of beta B2-crystallin and gamma-crystallins fold to form similar N- and C-terminal domains, the conformation of the connecting peptides are such that the gamma-crystallins are monomers and the beta-crystallin is a dimer. Unlike gamma-crystallins, the numerous beta-crystallins have extensions of variable sequence from the globular domains. We have tested the effect of removing the N- and C-terminal extensions from rat beta B2-crystallin using a bacterial expression system. Abundant proteins were produced in Escherichia coli using the pET or pQE vectors. Full-length and truncated proteins were purified and checked for refolding using circular dichroism. Sizing of the truncated proteins using gel filtration chromatography showed that the absence of either the N- or C-terminal extension does not affect dimerization of beta B2-crystallin.
Assuntos
Cristalinas/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Sequência de Bases , Dicroísmo Circular , Cristalinas/genética , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismoRESUMO
The three crystallins, alpha B-crystallin, tau-crystallin/alpha-enolase and epsilon-crystallin/lactate dehydrogenase B are all stress induced proteins and may thus share common regulatory elements. However, no evidence was found for coordinate expression of these three genes during duck lens development. The alpha B- and alpha-enolase/tau-crystallin mRNAs accumulate with similar kinetics between day 12 and day 24 of embryonic development but differ in their epithelial versus fibre cell location; the LDH-B/epsilon-crystallin transcript shares its preferential location in the fibre cell with alpha B-crystallin but differs in its developmental pattern of expression. The accumulation of LDH-B/epsilon-crystallin mRNA in heart and lens followed a similar developmental pattern. In contrast, the alpha-enolase/tau-crystallin mRNA level in heart decreases while the level in lens rises. The LDH-B gene is used as a crystallin gene in duck but not in chicken. This species-specific difference may correlate with the difference in LDH-B activity between various chicken and duck tissues: the retina and pancreas in duck have significantly higher LDH-B activity than in chick, while heart, muscle, stomach, liver, intestine and kidney all have much higher LDH-B activity in chicken than in duck.
Assuntos
Cristalinas/genética , Patos/embriologia , Coração/embriologia , Cristalino/embriologia , RNA Mensageiro/análise , Animais , Northern Blotting , Galinhas/metabolismo , Patos/genética , Patos/metabolismo , Epitélio/metabolismo , Regulação da Expressão Gênica , Isoenzimas , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Cristalino/metabolismo , Miocárdio/metabolismo , Fosfopiruvato Hidratase/genéticaRESUMO
The locus for the hereditary human Coppock-like cataract (CCL) is closely linked to a particular combination of polymorphic TaqI sites within the human gamma-crystallin gene cluster. Mapping of these sites shows that they define a 15 kb region encompassing the gamma D and psi gamma E gene. The gamma D and the psi gamma E gene were cloned from the CCL chromosome and characterized. The gamma D gene was functionally equivalent to its allele cloned from a wild-type chromosome. The CCL psi gamma E gene contains a cluster of sequence changes within and around its TATA box. Together these cause a ten-fold increase in the activity of the psi gamma E promoter, raising the level of expression of this gene to 30% of that of the gamma D gene. The predicted protein product of the psi gamma E gene is a 6 kD N-terminal gamma-crystallin fragment. Reactivation of the psi gamma E gene and concomitant overexpression of the gamma-crystallin fragment could be the cause of the Coppock-like cataract.
Assuntos
Catarata/genética , Cristalinas/genética , Pseudogenes , Sequência de Bases , Regulação da Expressão Gênica , Haplótipos/genética , Humanos , Dados de Sequência Molecular , Polimorfismo Genético , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , TATA BoxRESUMO
We have developed a sensitive and quantitative RT-PCR assay for the determination of tissue factor (TF) mRNA levels in human cells. An in vitro synthesized internal standard RNA was used to correct for differences in reverse transcription or amplification of various RNA samples. The PCR products were quantitated by hybridization. The sensitivity was such that less than 0.2 microgram of total endothelial RNA sufficed to measure its TF mRNA content. The RT-PCR assay was used to determine TF mRNA levels in endothelial cells treated with a factor from human melanoma cells and/or TNF. In this way the amount of TF mRNA could be induced to a level that was at least 80-fold higher than that in non-induced cells. This increase was in the same order of magnitude as the induction of measured TF activity.
Assuntos
Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/análise , RNA Mensageiro/genética , Tromboplastina/genética , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Endotélio Vascular/química , Endotélio Vascular/metabolismo , Estudos de Avaliação como Assunto , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/normas , Reação em Cadeia da Polimerase/estatística & dados numéricos , RNA Mensageiro/normas , Padrões de Referência , Sensibilidade e Especificidade , Tromboplastina/biossínteseRESUMO
The gene encoding the gametocyte/gamete-specific membrane protein Pfs48/45 of Plasmodium falciparum has been cloned. The Pfs48/45 gene is a non-interrupted, single copy gene that codes for a hydrophobic, non-repetitive protein of 448 amino acid residues containing a putative signal peptide at the N-terminus, a hydrophobic C-terminus and 7 potential N-glycosylation sites. Antibodies directed against a Pfs48/45-glutathione-S-transferase fusion protein reacted with both the 45-kDa and 48-kDa proteins of gametocytes. When Pfs48/45 is expressed in the baculovirus-insect cell system the recombinant Pfs48/45 protein is targeted and exposed to the insect cell surface in such a configuration that it is recognized by transmission-blocking anti-45/48-kDa monoclonal antibodies.
Assuntos
Antígenos de Protozoários/genética , Glicoproteínas de Membrana/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Antígenos de Protozoários/imunologia , Sequência de Bases , Northern Blotting , Western Blotting , Clonagem Molecular , DNA de Protozoário , Imunofluorescência , Dados de Sequência Molecular , Plasmodium falciparum/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
By screening of cDNA and genomic libraries of Plasmodium falciparum with a DNA probe derived from the cognate beta-tubulin gene, gene Pf gamma tub has been identified that codes for gamma-tubulin, a newly discovered member of the tubulin superfamily that is indispensible for nuclear division and microtubule assembly [12]. Gene Pf gamma tub is not interrupted by introns and only present as a single-copy in the parasite genome. Its encoded amino acid sequence (452 amino acids; M(r) 50,560) has a 63% similarity to the gamma-tubulins encoded by Aspergillus nidulans, Schizosaccharomyces pombe, Drosophila melanogaster, Xenopus laevis and Homo sapiens. This figure is significantly (approx. 8%) lower than the average identity between the gamma-tubulins of the latter five species suggesting that during evolution the genes have been exposed to different selection pressures. The identity of gamma-tubulin to the Plasmodium falciparum encoded alpha- and beta-tubulins is 30 and 33%, respectively.
Assuntos
Genes de Protozoários , Plasmodium falciparum/genética , Tubulina (Proteína)/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA de Protozoário/genética , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
In duck the single copy lactate dehydrogenase B (LDH-B) gene also encodes an abundant lens protein, epsilon-crystallin. The LDH-B/epsilon-crystallin gene consists of eight exons, of which the first is non-coding. The promoter region lacks a TATA box, is very GC-rich and contains multiple consensus Sp1 binding sites. The gene has two discrete transcription start sites located 28 base-pairs apart. Both sites are used about equally in heart tissue, while transcription from the downstream start site predominates in the lens. For maximal promoter activity in lens or heart, sequences from the first intron are required. The enhancer(s) in this intron is promoter specific as it could not activate the tk promoter. Studies at the RNA level show that the overexpression of the LDH-B/epsilon-crystallin gene in the lens is regulated at the transcriptional level, yet no tissue-specific regulatory elements could be detected in a region spanning from -1.9 kb (1 kb = 10(3) bases or base-pairs) up to the translation initiation site in the second exon. The basis for the differential expression of the LDH-B/epsilon-crystallin gene in duck heart and lens is the usage of the downstream transcription initiation site. However, our results do not allow a distinction between activation of the downstream transcription start site in the lens or repression of the use of this site in heart.
Assuntos
Cristalinas/genética , Regulação Enzimológica da Expressão Gênica , L-Lactato Desidrogenase/genética , Regiões Promotoras Genéticas , Animais , Composição de Bases , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , DNA/metabolismo , Patos , Elementos Facilitadores Genéticos , Íntrons , Isoenzimas , L-Lactato Desidrogenase/metabolismo , Metilação , Dados de Sequência Molecular , Miocárdio/metabolismo , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Rat lens nuclear extracts contain a factor that binds to position -57 to -46 of the rat gamma D-crystallin promoter region. This factor protects the sequence 5'-CTGCCAACGCAG-3' in a footprint analysis. Binding to this region is crucial for maximal promoter activity in rat lens cells, but this sequence was unable to act as an enhancer when cloned in front of a heterologous promoter. A region directly upstream from this activating sequence, between position -85 to -67, acts as a strong silencer of promoter activity in non-lens cells. This silencing effect is mediated by trans-acting factor(s). Our data provide evidence for two regulatory elements in rat gamma D-crystallin gene expression, an activating sequence active in lens cells and a silencing sequence active only in non-lens cells. The factor that binds to the activating sequence could be detected only in lens cells and may be a determinant of the lens-specific expression of the gamma-crystallin genes.
Assuntos
Cristalinas/genética , Cristalino/fisiologia , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , DNA/genética , DNA/isolamento & purificação , Epitélio/fisiologia , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleotídeos , Regiões Promotoras Genéticas , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por RestriçãoRESUMO
Explanted rat lens epithelial cells differentiate synchronously in vitro to lens fiber cells in the presence of basic fibroblast growth factor (bFGF). We have monitored the expression of the three rat crystallin gene families, the alpha-, beta-, and gamma-crystallin genes, during this process. The expression of these gene families is sequentially activated, first the alpha-crystallin genes at Day 1, then the beta-crystallin genes at Day 3, and finally the gamma-crystallin genes at Day 8. The steady state levels of alpha- and beta-crystallin mRNA are not affected by incubation with actinomycin D, suggesting that these mRNAs are stable. Nevertheless, all crystallin mRNAs disappear from the differentiated explants between Days 10 and 11, a process signaled by bFGF. At this time a novel abundant mRNA appears. Cloning and sequencing showed that this mRNA encoded aldose reductase. Our results suggest a novel model for the regulation of crystallin synthesis during lens cell differentiation: a gene pulse delivers a certain amount of stable mRNA, this mRNA is removed at a later stage of differentiation by a stage-specific breakdown mechanism. Each of these regulatory steps requires a signal from bFGF.
Assuntos
Cristalinas/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Cristalino/metabolismo , RNA Mensageiro/análise , Fatores Etários , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cristalinas/biossíntese , Cristalino/citologia , RNA Mensageiro/metabolismo , RatosRESUMO
This investigation describes how the binding characteristics of the single-stranded DNA-binding protein encoded by gene V of bacteriophage M13, are affected by single-site amino acid substitutions. The series of mutant proteins tested includes mutations in the purported monomer-monomer interaction region as well as mutations in the DNA-binding domain at positions which are thought to be functionally involved in monomer-monomer interaction or single-stranded DNA binding. The characteristics of the binding of the mutant proteins to the homopolynucleotides poly(dA), poly(dU) and poly(dT), were studied by means of fluorescence-titration experiments. The binding stoichiometry and fluorescence quenching of the mutant proteins are equal to, or lower than, the wild-type gene V protein values. In addition, all proteins measured bind a more-or-less co-operative manner to single-stranded DNA. The binding affinities for poly(dA) decrease in the following order: Y61H greater than wild-type greater than F68L and R16H greater than Y41F and Y41H greater than F73L greater than R21C greater than Y34H greater than G18D/Y56H. Possible explanations for the observed differences are discussed. The conservation of binding affinity, also for mutations in the single-stranded DNA-binding domain, suggests that the binding to homopolynucleotides is largely non-specific.
Assuntos
Bacteriófagos/química , Proteínas de Ligação a DNA/metabolismo , Polidesoxirribonucleotídeos/metabolismo , Proteínas Virais/metabolismo , Bacteriófagos/genética , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Genes Virais , Espectroscopia de Ressonância Magnética , Mutação , Conformação Proteica , Solubilidade , Espectrometria de Fluorescência , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Comparison of the partial sequences of the human beta B2-1- and beta B2-2-crystallin genes with orthologous rat or calf sequences shows that the fourth exon sequence of the human beta B2-2 gene contains a one triplet deletion and a mutated splice acceptor site. No transcripts from the beta B2-2-crystallin gene could be detected in the human lens. These data suggest that the human beta B2-2-crystallin gene is a pseudogene.