RESUMO
OBJECTIVE: To evaluate C-reactive protein at presentation and during hospitalisation in dogs with acute kidney injury resulting from leptospirosis to compare C-reactive protein at presentation in dogs with acute kidney injury of different aetiology and to study its correlation with markers of inflammation, azotaemia and survival. MATERIALS AND METHODS: Prospective observational study of 41 dogs with acute kidney injury secondary to leptospirosis and 15 control dogs with acute kidney injury of different aetiology. C-reactive protein was measured at presentation in both groups and daily for 7 days in a subgroup of 28 dogs with leptospirosis. The associations of C-reactive protein with neutrophil count, albumin, urea, creatinine and survival were analysed. RESULTS: C-reactive protein was increased at presentation in all dogs with leptospirosis but was not significantly different from dogs with acute kidney injury of different cause. It was associated with markers of inflammation (neutrophil count, albumin) but not with azotaemia (creatinine, urea). It decreased gradually from presentation to day 4, with significantly lower concentrations in survivors than non-survivors. Initial C-reactive protein was only weakly associated with outcome, but its average concentration from presentation to day 2 was more strongly associated. Absolute and relative changes in C-reactive protein during hospitalisation and creatinine at presentation were not associated with survival. CLINICAL SIGNIFICANCE: Serial assessment of C-reactive protein may improve outcome prediction in dogs with leptospirosis compared with a single measurement at presentation or with markers of renal function.
Assuntos
Injúria Renal Aguda/veterinária , Doenças do Cão , Leptospirose/veterinária , Animais , Proteína C-Reativa , Cães , Cinética , Prognóstico , Estudos ProspectivosRESUMO
The promoters of two interferon-induced genes (the ISG54 and guanylate-binding protein [GBP] genes) have been analyzed in whole cells and in isolated nuclei by using a new genomic sequencing technique. The ISG54 gene contains an interferon-simulating response element (ISRE), earlier shown to be necessary and sufficient for alpha interferon (IFN-alpha) induction, that appeared complexed with proteins in both transcribing and nontranscribing cells. However, the extent of protection and hypersensitivity to DNase I or dimethyl sulfate within the ISRE region was changed upon transcriptional induction, suggesting the binding of different factors in different transcriptional states. In addition to the ISRE, the GBP gene needs a newly recognized DNA element, called the GAS, that partly overlaps the ISRE for full induction by either IFN-alpha or IFN-gamma. This GAS element was transiently protected against DNase I in the nuclei of interferon-treated cells but was not protected at later times when transcription reached maximal levels. Thus, the GAS-binding activity may be necessary only transiently for the initial assembly of a transcription initiation complex on the GBP promoter. Dimethyl sulfate methylation of genomic DNA performed on intact cells showed a characteristic sensitivity over the GAS that correlated with transcription levels and that persisted longer than did DNase I protection over the GAS. These results demonstrate the involvement of the GAS in IFN-alpha and -gamma induction of GBP and suggest the presence of an altered DNA conformation or a small protein in the major groove of the GAS associated with ongoing GBP transcription.
Assuntos
Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Interferon-alfa/fisiologia , Interferon gama/fisiologia , Transcrição Gênica , Sequência de Bases , Linhagem Celular , DNA , Desoxirribonuclease I/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Ésteres do Ácido Sulfúrico/farmacologia , TATA BoxRESUMO
Hepatocyte nuclear factor 4 (HNF-4) is a liver-enriched transcription factor and a member of the steroid hormone receptor superfamily. HNF-4 is required for the hepatoma-specific expression of HNF-1 alpha, another liver-enriched transcription factor, suggesting the early participation of HNF-4 in development. To prepare for further study of HNF-4 in development, the tissue-specific expression of the mouse HNF-4 gene was studied by analyzing the promoter region for required DNA elements. DNase-hypersensitive sites in the gene in liver and kidney tissues were found in regions both distal and proximal to the RNA start that were absent in tissues in which HNF-4 expression did not occur. By use of reporter constructs in transient-transfection assays and with transgenic mice, a region sufficient to drive liver-specific expression of HNF-4 was identified. While an HNF-1 binding site between bp -98 and -68 played an important role in the hepatoma-specific promoter activity of HNF-4 in transient-transfection assays, it was not sufficient for the liver-specific expression of a reporter gene in transgenic mice. Distal enhancer elements indicated by the presence of DNase I-hypersensitive sites at kb -5.5 and -6.5, while not functional in transient-transfection assays, were required for the correct expression of the mouse HNF-4 gene in animals.
Assuntos
Proteínas de Ligação a DNA , Fosfoproteínas , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Sítios de Ligação , Sondas de DNA/genética , Desoxirribonuclease I , Regulação da Expressão Gênica , Fator 4 Nuclear de Hepatócito , Rim/metabolismo , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA/genética , Distribuição Tecidual , Fatores de Transcrição/metabolismo , Transfecção , Células Tumorais Cultivadas/metabolismoRESUMO
The assembly of an RNA polymerase II initiation complex at a promoter is associated with the melting of the DNA template to allow the polymerase to read the DNA sequence and synthesize the corresponding RNA. Using the specific single-stranded modifying reagent KMnO4 and a new genomic sequencing technique, we have explored the melted regions of specific genes in genomic DNA of whole cells or of isolated nuclei. We have demonstrated for the first time in vivo the melting in the promoter proximal transcribed region that is associated with the presence of RNA polymerase II complexes. An interferon-inducible gene, ISG-54, exhibited KMnO4 sensitivity over approximately 300 nucleotides downstream of the RNA initiation site in interferon-treated cells when the gene was actively transcribed but not in untreated cells where the gene was not transcribed. The extent of KMnO4 modification was proportional to transcription levels. The KMnO4 sensitivity was retained when nuclei were isolated from induced cells but was lost if the engaged polymerases were further allowed to elongate the nascent RNA chains ("run-on"). The sensitivity to KMnO4 in isolated nuclei was retained if the run-on incubation was performed in the presence of alpha-amanitin, which blocks progress of engaged polymerases. A similar analysis identified an open sequence of only approximately 30 bases just downstream of the start site of the transthyretin (TTR) gene in nuclei isolated from mouse liver, a tissue where TTR is actively transcribed. This abrupt boundary of KMnO4 sensitivity, which was removed completely by allowing engaged polymerases to elongate RNA chains, suggests that most polymerases transcribing this gene paused at about position +20. The possibility of mapping at the nucleotide level the position of actively transcribing RNA polymerases in whole cells or isolated nuclei opens new prospects in the study of transcription initiation and elongation.
Assuntos
RNA Polimerase II/genética , Animais , Núcleo Celular/enzimologia , Mapeamento Cromossômico , DNA de Cadeia Simples/genética , Células HeLa , Humanos , Indicadores e Reagentes , Fígado/metabolismo , Camundongos , Conformação de Ácido Nucleico , Permanganato de Potássio , Pré-Albumina/genética , RNA/química , RNA/genética , RNA Polimerase II/metabolismo , Transcrição GênicaRESUMO
Dendritic cells (DC) are important cells at the interface between innate and adaptive immunity. DC have a key role in antigen processing and presentation to T cells. Effector functions of DC related to innate immunity have not been explored extensively. We show that bovine monocyte-derived DC (mDC) express inducible nitric oxide synthase (iNOS) mRNA and protein and produce NO upon triggering with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes (HKLM). An immunocytochemical analysis revealed that a sizeable subset (20-60%) copiously expresses iNOS (iNOShi) upon IFN-gamma/HKLM triggering, whereas the other subset expressed low levels of iNOS (iNOSlo). Monocyte-derived macrophages (mMphi) are more homogeneous with regard to iNOS expression. The number of cells within the iNOSlo mDC subset is considerably larger than the number of dead cells or cells unresponsive to IFN-gamma/HKLM. The large majority of cells translocated p65 to the nucleus upon triggering by IFN-gamma/HKLM. A contamination of mDC with iNOS-expressing mMphi was excluded as follows. (i) Cell surface marker analysis suggested that mDC were relatively homogeneous, and no evidence for a contaminating subset expressing macrophage markers (e.g. high levels of CD14) was obtained. (ii) iNOS expression was stronger in iNOShi mDC than in mMphi. The use of maturation-promoting stimuli revealed only subtle phenotypic differences between immature and mature DC in cattle. Nevertheless, these stimuli promoted development of considerably fewer iNOShi mDC upon triggering with IFN-gamma/HKLM. Immunocytochemical results showed that although a significant proportion of cells expressed iNOS only or TNF only upon triggering with IFN-gamma/HKLM, a significant number of cells expressed both iNOS and TNF, suggesting that TNF and iNOS producing (TIP) DC are present within bovine mDC populations obtained in vitro.
Assuntos
Bovinos/imunologia , Células Dendríticas/enzimologia , Imunidade Inata/imunologia , Óxido Nítrico Sintase Tipo II/biossíntese , Animais , Western Blotting/veterinária , Células Dendríticas/citologia , Células Dendríticas/imunologia , Citometria de Fluxo/veterinária , Imuno-Histoquímica/veterinária , Interferon gama/imunologia , Listeria monocytogenes/imunologia , Ativação Linfocitária/imunologia , Macrófagos/citologia , Macrófagos/enzimologia , Macrófagos/imunologia , Óxido Nítrico Sintase Tipo II/genética , RNA/química , RNA/genética , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The molecular mechanisms underlying transcription elongation and their role in gene regulation are poorly characterized in eukaryotes. A number of genes, however, have been proposed to be regulated at the level of transcription elongation, including c-myc, c-fos and c-myb. Here, we analyze the control of transcription elongation at the mouse c-fos gene at the nucleotide level in intact cells. We find that RNA polymerases are engaged in the promoter-proximal part of the gene in the absence of gene activation signals and mRNA synthesis. Importantly, we determine that the engaged RNA polymerases originate from a continuous initiation of transcription which, in the absence of gene activation signals, terminate close to the promoter. We also observe that the c-fos gene presents an active chromatin conformation, with the promoter and upstream regulatory sequences constitutively occupied by proteins, accounting for the continuous initiation of RNA polymerase complexes. We propose that activation of c-fos gene expression results primarily from the assembly of elongation-competent RNA polymerases that can transcribe the complete gene. Our results suggest that the engaged RNA polymerases found downstream of a number of other eukaryotic promoters may be associated with transcription termination of elongation-incompetent polymerases in the absence of activating signals.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes fos/genética , Células 3T3 , Animais , Pegada de DNA , Camundongos , Transcrição Gênica , Ativação TranscricionalRESUMO
We have mapped the DNA sequences bound to the nuclear scaffold along 320,000 base-pairs of a genetically well-defined region of the Drosophila chromosome. We have found that the domains delimited by the scaffold attachment regions are heterogeneous in size (ranging from 26,000 to 112,000 base-pairs in this interval), and that the attachment sites are within unique sequences as judged by blot hybridization. We also found that looped domains contain up to five, or even eight, unrelated genes including, in some cases, more than one transcribed gene. The loop organization unravelled here in cultured cells does not correspond to the banding pattern seen in salivary gland polytene chromosomes.
Assuntos
Cromossomos , DNA/genética , Drosophila melanogaster/genética , Genes , Animais , Sequência de Bases , Bandeamento Cromossômico , Cromossomos/ultraestruturaRESUMO
We have examined the higher-order loop organization of DNA in interphase nuclei and metaphase chromosomes from Drosophila Kc cells, and we detect no changes in the distribution of scaffold-attached regions (SARs) between these two phases of the cell cycle. The SARs, previously defined from experiments with interphase nuclei, not only are bound to the metaphase scaffold when endogenous DNA is probed but also rebind specifically to metaphase scaffolds when added exogenously as cloned, end-labeled fragments. Since metaphase scaffolds have a simpler protein pattern than interphase nuclear scaffolds, and both have a similar binding capacity, it appears that the population of proteins required for the specific scaffold-DNA interaction is limited to those found in metaphase scaffolds. Surprisingly, metaphase scaffolds isolated from Drosophila Kc cells contain both the lamin protein and a pore-complex protein, glycoprotein (gp) 188. To study whether lamin contributes to the SAR-scaffold interaction, we have carried out comparative binding studies with scaffolds from HeLa metaphase chromosomes, which are free of lamina, and from HeLa interphase nuclei. All Drosophila SAR fragments tested bind with excellent specificity to HeLa interphase scaffolds, whereas a subset of them bind to HeLa metaphase scaffolds. The maintenance of the scaffold-DNA interaction in metaphase indicates that lamin proteins are not involved in the attachment site for at least a subset of Drosophila SARs. This evolutionary and cell-cycle conservation of scaffold binding sites is consistent with a fundamental role for these fragments in the organization of the genome into looped domains.
Assuntos
Cromossomos/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Animais , Drosophila melanogaster , Células HeLa , Humanos , Laminas , MetáfaseRESUMO
We have previously identified a number of specific DNA fragments called SARs (scaffold-associated regions) that are associated with the nuclear scaffold and define the basis of DNA loops. We demonstrate that cloned DNA fragments containing SAR sequences bind to nuclear scaffolds in vitro with the same specificity as have genomic SAR fragments. This specific interaction is observed with the biochemically complex type I scaffolds. These scaffolds are composed of the nuclear lamina proteins and a set of other proteins that forms the internal network of these structures. So-called type II scaffolds, which are composed primarily of the lamina proteins and lack the proteins of the internal network, do not bind the SAR fragments at a detectable level. Competition experiments show that different SARs share common structural elements and can bind to the same sites on the nuclear scaffold, although with different affinities. Moreover, the SAR binding sites appear to be evolutionarily conserved, as all the Drosophila SARs also bind with identical specificity to nuclear scaffolds derived from rat liver nuclei. These Sar interaction studies were carried out with lithium 3,5-diiodosalicylate-extracted nuclei. Interestingly, scaffolds prepared by high-salt extraction also bind the genomic and exogenously added SAR fragments specifically. However, the endogenous transcribed sequences, as opposed to the same fragments added as purified DNA, associate randomly with these scaffolds.
Assuntos
DNA/metabolismo , Proteínas Nucleares/metabolismo , Animais , Sítios de Ligação , Evolução Biológica , Mapeamento Cromossômico , Drosophila melanogaster , Técnicas In Vitro , Conformação de Ácido NucleicoRESUMO
Hepatocyte growth factor/scatter factor (HGF/SF) is a recently characterised molecule with many remarkable functions. Its involvement in important processes such as cell proliferation, cell migration, morphogenesis and organ development implies that its activity should be tightly regulated. To understand the molecular mechanisms controlling HGF/SF transcription, we have analysed DNaseI hypersensitive sites (DHS) along rat and human HGF/SF genes in various tissues and cell types. We identified five DHS along the rat gene, two in the 5'-flanking region and three in the first intron. These sites are only found in rat tissues and rat cell lines, which express HGF/SF. The strongest hypersensitive site map to a region that corresponds to the promoter by start site analysis. A single tissue-specific DHS is present in human cell lines that express HGF/SF and corresponds to the promoter region. Our results suggest that chromatin accessibility plays a major role in the regulation of HGF/SF transcription regulation.
Assuntos
Cromatina/genética , Fator de Crescimento de Hepatócito/genética , Interleucina-3/química , Regiões Promotoras Genéticas/genética , Animais , Linhagem Celular , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Humanos , Nuclease do Micrococo/metabolismo , RNA Mensageiro/metabolismo , Ratos , Transcrição Gênica/genéticaRESUMO
Chromatin architecture plays a decisive role in many aspects of transcription regulation. We have tested the role of specific chromatin structures in c-fos gene regulation, using a gene transfer system based on episomes derived from the Epstein-Barr virus (EBV). This system reproduces in several respects the chromatin structure and regulation of the chromosomal c-fos gene. Using this approach, we first demonstrate that the pausing of RNA polymerase II downstream of the transcriptional start site does not require precisely positioned nucleosomes. Indeed, changing the pattern of MNase hypersensitive sites along the transcribed sequence does not perturb RNA polymerase II pausing or the regulation of the c-fos gene. Next, we show that a putative nucleosome positioned between the SIE/SRE elements (-300) and the CRE/TATA elements (-36) is not necessary for activation by a variety of inducers. Accordingly, total or partial deletion of the putative nucleosome sequence does not disturb c-fos regulation while the two regulatory sites flanking the nucleosome sequence remain hypersensitive to MNase. As described in this paper, EBV episomes are useful vectors to critically examine the role of the chromatin structure in gene transcription for human cells.
Assuntos
Nucleossomos/genética , Proteínas Proto-Oncogênicas c-fos/genética , Animais , Linhagem Celular , Cromatina/genética , Colforsina/farmacologia , DNA/genética , DNA/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 4/genética , Humanos , Ionomicina/farmacologia , Células Jurkat , Luciferases/genética , Luciferases/metabolismo , Camundongos , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
Transcription elongation regulates c-fos expression in mouse and human cells. In the inactive state of the gene, RNA polymerases are engaged only in the promoter-proximal region. Upon activation, RNA polymerases move efficiently along the complete gene. We have used Epstein-Barr virus (EBV) episomes as a gene transfer system to study the role of promoter-proximal pausing and transcript elongation in c-fos expression. We find that the sequence located immediately downstream of the transcriptional start site specifies pausing of RNA polymerases, dependent on both its orientation and position relative to the promoter. This sequence is, however, not necessary to maintain repression in the absence of a stimulus. As promoter-proximal pausing is therefore not a repression mechanism for the c-fos gene, the promoter and enhancer sequences are the main determinants of RNA polymerase elongation competence. Surprisingly, we find that promoter-proximal pausing further increases transcriptional levels from a variety of promoters. These observations lead us to hypothesize that promoter-proximal pausing of RNA polymerase II augments c-fos expression by allowing more efficient phosphorylation of the C-terminal domain of the large subunit.
Assuntos
Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-fos/genética , RNA Polimerase II/metabolismo , Animais , Linhagem Celular , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 4/genética , Humanos , Células Jurkat , Luciferases/genética , Luciferases/metabolismo , Camundongos , Plasmídeos/genética , RNA/efeitos dos fármacos , RNA/genética , RNA/metabolismo , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Regulation of transcription is a key feature of the HIV-1 life cycle. In recent years, various sequence elements and transcription factors have been shown to participate in HIV-1 transcription control. New evidence, however, has shown that chromatin organization plays a key role in the establishment of a transcriptionally regulated HIV-1 LTR. The present review discusses recent data obtained on reconstituted and genomic HIV-1 chromatin templates.
Assuntos
Cromatina , HIV-1/genética , Transcrição Gênica , Animais , Pegada de DNA , Desoxirribonuclease I/metabolismo , Previsões , Regulação Viral da Expressão Gênica , Produtos do Gene tat/metabolismo , Repetição Terminal Longa de HIV , Humanos , Nucleossomos , Elongação Traducional da Cadeia Peptídica , Proteínas/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência HumanaAssuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Genoma , Animais , Fracionamento Celular/métodos , Núcleo Celular/ultraestrutura , Células Cultivadas , DNA/isolamento & purificação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Desoxirribonuclease I , Células HeLa , Humanos , Indicadores e Reagentes , Leucócitos/citologia , Leucócitos/metabolismo , Mutagênese , Mutagênicos/farmacologia , Reação em Cadeia da Polimerase/métodos , Permanganato de Potássio/farmacologia , Sondas RNA/síntese química , Ésteres do Ácido Sulfúrico/farmacologia , Moldes GenéticosRESUMO
In vivo examination of the occupancy of DNA elements that can regulate transcription is critical to reveal which proteins actually take part in establishing and maintaining gene expression. We describe a new genomic sequencing method involving the rapid purification of relevant DNA segments from the bulk of the genomic DNA using a biotinylated riboprobe. The purified sequences are revealed by a single primer extension using Taq DNA polymerase. We used this technique to study the promoter and the enhancer of mouse transthyretin (TTR), a gene highly expressed in the liver. Footprints showed high liver-specific occupancy of some, but not all, of the DNA sites that had been identified as important for expression by transfection studies in hepatoma cells. In addition, several previously undetected sites were observed that bound proteins specifically in liver. These results suggest that not all demonstrable binding sites are involved in ongoing transcription and that in vivo studies may reveal additional and probably more relevant sites.
Assuntos
Proteínas de Ligação a DNA/química , Fígado/química , Pré-Albumina/genética , Animais , Impressões Digitais de DNA , Eletroforese em Gel de Ágar , Camundongos , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
The expression of genes in the liver is mostly controlled at the transcriptional level and depends on the regulatory interactions between cis-acting sequences and trans-acting molecules. Proximal promoters and distant enhancers in combination with a number of hepatocyte-enriched DNA-binding proteins and general transcription factors interact specifically with these elements and control the expression of liver-specific genes. Hepatocyte-enriched regulatory proteins have been isolated from liver nuclear extracts, characterized, and their corresponding genes have been cloned. These include the hepatocyte nuclear factors 1, 3, 4 (HNF-1,3,4), some members of the CAAAT/enhancer binding protein (C/EBP) family, and D site binding protein (DBP). These factors belong to larger families and are able to form heterodimers, perhaps with the exception of the HNF-3 family, with other members of the same family. Interestingly, the majority of the genes encoding such proteins are themselves regulated at the transcriptional level, although both transcriptional and post-transcriptional events modulate their expression during development, hepatocyte differentiation and disease, suggesting that a transcriptional cascade may play a critical role in mammalian liver development and differentiation.
Assuntos
Regulação da Expressão Gênica , Hepatopatias/genética , Fígado/metabolismo , Animais , Sequência de Bases , Diferenciação Celular/genética , Fígado/citologia , Fígado/crescimento & desenvolvimento , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , RoedoresRESUMO
Data are presented for sequence-specific chromatin-loop organization in histone-depleted nuclei from Drosophila melanogaster Kc cells. We find one loop for each of the tandemly repeated histone gene clusters. The attachment site is localized in the A + T rich H1-H3 spacer on a 657 bp fragment. In the cluster of the hsp70 heat-shock genes, in both control and heat-shocked cells, we find two attachment sites in close proximity upstream of regulatory elements. The transcribed sequences are not associated with the nuclear scaffold in control or in heat-shocked cells. A family of attachment sites related by hybridization to those of the hsp70 genes was discovered.
Assuntos
Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , DNA/isolamento & purificação , Genes , Histonas/genética , Animais , Linhagem Celular , Cromatina/metabolismo , Enzimas de Restrição do DNA , Drosophila melanogaster , Histonas/isolamento & purificaçãoRESUMO
We have been able to map specific DNA fragments at the bases of chromatin loops with the help of a novel extraction procedure by using lithium-3',5'-diiodosalicylate. One such scaffold-attached region (SAR) is found in the non-transcribed spacer in each repeat of the histone gene cluster, on a 657 base pair (b.p.) restriction fragment. Exonuclease III digestion has localized two protein-binding domains on the SAR of the histone cluster. Each covers approximately 200 b.p. and they are separated by a nuclease-accessible region of about 100 b.p. These domains are rich in sequences closely related to the topoisomerase II cleavage consensus. We have studied the scaffold association of three developmentally regulated genes of Drosophila melanogaster: alcohol dehydrogenase (Adh), the homoeotic gene fushi tarazu (ftz) and Sgs-4, a gene encoding one of the glue proteins secreted by third-instar larvae. We find regions attached to the nuclear scaffold (SARS) both 5' and 3' of all three genes, defining small domains ranging from 4.5 to 13 kilobases. In the case of Adh, a gene with two promoters, we find two upstream and two downstream SARS. Those 5' of the gene co-map with regulatory regions for the adult and the larval transcripts, respectively. For Sgs-4, the 5' SAR covers 866 b.p. immediately upstream of the transcript, and encompasses the 200 b.p. regulatory region defined by two deletion mutants that produce little or no Sgs-4 protein. In ftz the 5' SAR is found 4.8 kilobases upstream of the start of transcription within a 2.5 kilobase element required for a high level of ftz expression in the early embryo. Sequence analysis of five upstream SARS reveals clusters of sequences closely related to the cleavage consensus of topoisomerase II. In addition, they contain multiple copies of two sequence motifs: a specific 10 b.p. A-rich sequence, and another 10 b.p. T-rich stretch. In conclusion, the intimate association of the SAR with the upstream/enhancer elements, the presence of clustered sequences highly homologous to the topoisomerase II cleavage consensus, and the localization of topoisomerase II in the scaffold, suggest a structure-function relation between chromosome organization and gene expression.
Assuntos
Cromossomos/fisiologia , Genes , Transcrição Gênica , Animais , Cromossomos/ultraestrutura , Genes Reguladores , Regiões Promotoras GenéticasRESUMO
The mouse gene Cebp, encoding the DNA-binding protein C/EBP, has been localized to the proximal region of chromosome 7 by determining the strain distribution patterns of a restriction fragment length polymorphism among the BXD and AKXL recombinant inbred mouse lines.