RESUMO
We analyzed the pattern of spindle microtubule (MT) regrowth after cold- or colcemid-induced MT depolymerization in Drosophila S2 cells. Cold-induced MT disassembly at low temperature (2 °C) destroyed kinetochore-driven MT regrowth without affecting astral MT formation. Conversely, colcemid-induced MT depolymerization strongly impaired centrosome-dependent MT nucleation, allowing kinetochore-driven MT regrowth. Collectively, these results indicate that the kinetochore- and the centrosome-mediated MT assembly pathways exploit molecular mechanisms that are at least in part different.
Assuntos
Proteínas de Drosophila/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Microtúbulos/genética , Fuso Acromático/genética , Tubulina (Proteína)/genéticaRESUMO
Mammalian housekeeping promoters represent a class of regulatory elements different from those of tissues-specific genes, lacking a TATA box and associated with CG-rich DNA. We have compared the organization of the housekeeping Htf9 promoter in different cell types by genomic footprinting. The sites of in vivo occupancy clearly reflected local combinations of tissue-specific and ubiquitous binding factors. The flexibility of the Htf9 promoter in acting as the target of cell-specific combinations of factors may ensure ubiquitous expression of the Htf9-associated genes.
Assuntos
DNA/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Células 3T3/química , Animais , Sequência de Bases , Sítios de Ligação , Carcinoma Hepatocelular/química , Linhagem Celular , Núcleo Celular/química , Embrião de Mamíferos , Fibroblastos/química , Humanos , Neoplasias Hepáticas/química , Neoplasias Hepáticas Experimentais/química , Camundongos , Dados de Sequência Molecular , Neuroblastoma/química , Reação em Cadeia da Polimerase , Ratos , Sequências Repetitivas de Ácido Nucleico , Células-Tronco/química , TATA Box , Células Tumorais CultivadasRESUMO
The mouse CpG island HTF9 harbours a bidirectional promoter shared by two housekeeping genes that are arranged head-to-head. We have previously identified several protein binding-elements across the CpG island, yet a short region around the initiation region was found to be capable of bidirectional transcription in transient expression assays, suggesting that the multiple elements of the HTF9 promoter are functionally redundant. We have now compared the binding activities in nuclear extracts from different cell types. Two protein-binding elements of HTF9 interact with widely distributed factors. A potentially strong Sp1 binding site was also identified, however Sp1 appeared to bind efficiently to its target sequence with extracts prepared from proliferating cultured cells, but not from adult organs. On the other hand, the CCAAT box upstream of one gene (HTF9-A) interacted with a liver-enriched factor, whereas no binding was detected with cultured fibroblasts extracts. Consistently, deletion of the CCAAT box affected transient expression from the HTF9-A promoter in hepatocyte, but not in fibroblast, cultures. Our results suggest that ubiquitous expression of housekeeping promoters results from the activation of alternative elements in different cell types.
Assuntos
Regiões Promotoras Genéticas/fisiologia , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica/fisiologia , Animais , Sequência de Bases , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Fosfatos de Dinucleosídeos/genética , Genes/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
The mouse HTF9 locus contains two genes that are bidirectionally transcribed with opposite polarity from a shared CpG-rich island. Both genes were previously shown to be expressed in a housekeeping fashion in mouse. We have now determined the molecular organization of the genes over 12 kb surrounding the island. In addition, we show that the HTF9 locus resides in the proximal region of mouse chromosome 16. We have sequenced the cDNAs corresponding to both divergent transcripts. Both genes appear to code for novel proteins that are structurally unrelated to each other. Finally, we show that both genes are highly conserved and efficiently expressed in human cells.
Assuntos
Homologia de Genes/genética , Proteínas Nucleares , Regiões Promotoras Genéticas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Proteína ran de Ligação ao GTP , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Cruzamentos Genéticos , Éxons/genética , Expressão Gênica/fisiologia , Variação Genética/genética , Humanos , Íntrons/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas/genética , Proteínas de Ligação a RNARESUMO
The mouse CpG-rich island HTF9 harbours the divergent RNA initiation sites shared by two genes that are both expressed in a housekeeping fashion. In this work we have analyzed the architecture of the HTF9 promoter. Gel shift assays were first employed to locate nuclear factor-binding sites within HTF9. Multiple protein-binding sites were identified across a 500 bp-long region, two of which appear to interact with novel factors. Deletion analysis was used to determine the requirements for the different sites in transient expression of a CAT reporter gene. Although multiple elements contributed to the overall promoter strength in each orientation, extensive deletions failed to affect the basal level of transcription from HTF9 in either direction. Thus, only a subset of elements is necessary to activate transcription from HTF9. Functional redundancy may be a general feature of housekeeping CpG-rich promoters.