RESUMO
SUN-domain proteins interact directly with KASH-domain proteins to form protein complexes that connect the nucleus to every major cytoskeleton network. SUN-KASH protein complexes are also required for attaching centrosomes to the nuclear periphery and for alignment of homologous chromosomes, their pairing and recombination in meiosis. Other functions that require SUN-domain proteins include the regulation of apoptosis and maturation and survival of the germline. Laminopathic diseases affect the distribution of the SUN-KASH complexes, and mutations in KASH-domain proteins can cause Emery Dreifuss muscular dystrophy and recessive cerebellar ataxia. This review describes our current knowledge of the role of SUN-KASH domain protein complexes during development, meiosis and disease.
Assuntos
Núcleo Celular/metabolismo , Laminas/fisiologia , Meiose/fisiologia , Proteínas de Membrana/fisiologia , Motivos de Aminoácidos/fisiologia , Animais , Apoptose/fisiologia , Centrossomo/metabolismo , Ataxia Cerebelar/genética , Cromossomos/metabolismo , Citoesqueleto/metabolismo , Humanos , Laminas/química , Laminas/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Biológicos , Distrofia Muscular de Emery-Dreifuss/genéticaRESUMO
The role of the Drosophila lamin protein in nuclear envelope assembly was studied using a Drosophila in vitro assembly system that reconstitutes nuclei from added sperm chromatin or naked DNA. Upon incubation of the embryonic assembly extract with anti-Drosophila lamin antibodies, the attachment of nuclear membrane vesicles to chromatin surface and nuclear envelope formation did not occur. Lamina assembly and nuclear membrane vesicles attachment to the chromatin were inhibited only when the activity of the 75-kD lamin isoform was inhibited in both soluble and membrane-vesicles fractions. Incubation of decondensed sperm chromatin with an extract that was depleted of nuclear membranes revealed the presence of lamin molecules on the chromatin periphery. In addition, high concentrations of bacterially expressed lamin molecules added to the extract, were able to associate with the chromatin periphery, and did not inhibit nuclear envelope assembly. After nuclear reconstitution, a fraction of the lamin pool was converted into the typical 74- and 76-kD isoforms. Together, these data strongly support an essential role of the lamina in nuclear envelope assembly.
Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Animais , Núcleo Celular/ultraestrutura , Sistema Livre de Células , Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Imunofluorescência , Laminas , Microscopia Eletrônica , Membrana Nuclear/ultraestruturaRESUMO
Two major immunocross-reactive polypeptides of the Drosophila nuclear envelope, distinguishable in interphase cells on the basis of one-dimensional SDS-PAGE mobility, have been localized to the nuclear lamina by immunoelectron microscopy. These have been designated lamins Dm1 and Dm2. Both lamins are apparently derived posttranslationally from a single, primary translation product, lamin Dm0. A pathway has been established whereby lamin Dm0 is processed almost immediately upon synthesis in the cytoplasm to lamin Dm1. Processing occurs posttranslationally, is apparently proteolytic, and has been reconstituted from cell-free extracts in vitro. Processing in vitro is ATP dependent. Once assembled into the nuclear envelope, a portion of lamin Dm1 is converted into lamin Dm2 by differential phosphorylation. Throughout most stages of development and in Schneider 2 tissue culture cells, both lamin isoforms are present in approximately equal abundance. However, during heat shock, lamin Dm2 is converted nearly quantitatively into lamin Dm1. Implications for understanding the regulation of nuclear lamina plasticity through normal growth and in response to heat shock are discussed.
Assuntos
Proteínas de Drosophila , Drosophila/embriologia , Membrana Nuclear/metabolismo , Nucleoproteínas/metabolismo , Animais , Clonagem Molecular , Drosophila/fisiologia , Embrião não Mamífero , Proteínas de Choque Térmico/genética , Temperatura Alta , Laminas , Membrana Nuclear/ultraestrutura , Nucleoproteínas/biossíntese , Nucleoproteínas/genética , Biossíntese de Proteínas , Transcrição GênicaRESUMO
Using a computer-based system for model building and analysis, three-dimensional models of 24 Drosophila melanogaster salivary gland nuclei have been constructed from optically or physically sectioned glands, allowing several generalizations about chromosome folding and packaging in these nuclei. First and most surprising, the prominent coiling of the chromosomes is strongly chiral, with right-handed gyres predominating. Second, high frequency appositions between certain loci and the nuclear envelope appear almost exclusively at positions of intercalary heterochromatin; in addition, the chromocenter is always apposed to the envelope. Third, chromosomes are invariably separated into mutually exclusive spatial domains while usually extending across the nucleus in a polarized (Rabl) orientation. Fourth, the arms of each autosome are almost always juxtaposed, but no other relative arm positions are strongly favored. Finally, despite these nonrandom structural features, each chromosome is found to fold into a wide variety of different configurations. In addition, a set of nuclei has been analyzed in which the normally aggregrated centromeric regions of the chromosomes are located far apart from one another. These nuclei have the same architectural motifs seen in normal nuclei. This implies that such characteristics as separate chromosome domains and specific chromosome-nuclear envelope contacts are largely independent of the relative placement of the different chromosomes within the nucleus.
Assuntos
Cromossomos/ultraestrutura , Drosophila melanogaster/ultraestrutura , Animais , Núcleo Celular/ultraestrutura , Membrana Nuclear/ultraestrutura , Glândulas Salivares/ultraestruturaRESUMO
A cDNA clone encoding a portion of Drosophila nuclear lamins Dm1 and Dm2 has been identified by screening a lambda-gt11 cDNA expression library using Drosophila lamin-specific monoclonal antibodies. Two different developmentally regulated mRNA species were identified by Northern blot analysis using the initial cDNA as a probe, and full-length cDNA clones, apparently corresponding to each message, have been isolated. In vitro transcription of both full-length cDNA clones in a pT7 transcription vector followed by in vitro translation in wheat germ lysate suggests that both clones encode lamin Dm0, the polypeptide precursor of lamins Dm1 and Dm2. Nucleotide sequence analyses confirm the impression that both cDNA clones code for the identical polypeptide, which is highly homologous with human lamins A and C as well as with mammalian intermediate filament proteins. The two clones differ in their 3'-untranslated regions. In situ hybridization of lamin cDNA clones to Drosophila polytene chromosomes shows only a single locus of hybridization at or near position 25F on the left arm of chromosome 2. Southern blot analyses of genomic DNA are consistent with the notion that a single or only a few highly similar genes encoding Drosophila nuclear lamin Dm0 exist in the genome.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Genes , Proteínas Nucleares/genética , Precursores de Proteínas/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Feminino , Imunoensaio , Laminas , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The Caenorhabditis elegans Bcl-2-like protein CED-9 prevents programmed cell death by antagonizing the Apaf-1-like cell-death activator CED-4. Endogenous CED-9 and CED-4 proteins localized to mitochondria in wild-type embryos, in which most cells survive. By contrast, in embryos in which cells had been induced to die, CED-4 assumed a perinuclear localization. CED-4 translocation induced by the cell-death activator EGL-1 was blocked by a gain-of-function mutation in ced-9 but was not dependent on ced-3 function, suggesting that CED-4 translocation precedes caspase activation and the execution phase of programmed cell death. Thus, a change in the subcellular localization of CED-4 may drive programmed cell death.
Assuntos
Apoptose , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Caspases , Proteínas de Helminto/metabolismo , Membrana Nuclear/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas Reguladoras de Apoptose , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Ligação ao Cálcio/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Genes de Helmintos , Proteínas de Helminto/genética , Imuno-Histoquímica , Mitocôndrias/metabolismo , Mutação , Fenótipo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
The number and complexity of genes encoding nuclear lamina proteins has increased during metazoan evolution. Emerging evidence reveals that transcriptional repressors such as the retinoblastoma protein, and apoptotic regulators such as CED-4, have functional and dynamic interactions with the lamina. The discovery that mutations in nuclear lamina proteins cause heritable tissue-specific diseases, including Emery-Dreifuss muscular dystrophy, is prompting a fresh look at the nuclear lamina to devise models that can account for its diverse functions and dynamics, and to understand its enigmatic structure.
Assuntos
Apoptose/genética , Estruturas do Núcleo Celular , Evolução Molecular , Proteínas Nucleares/fisiologia , Transcrição Gênica , Animais , Estruturas do Núcleo Celular/genética , Estruturas do Núcleo Celular/metabolismo , Eucariotos/fisiologia , Regulação da Expressão Gênica , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Heterocromatina/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Laminas , Distrofia Muscular de Emery-Dreifuss/genéticaRESUMO
The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events. Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions. Lamin, otefin, and YA are the three Drosophila nuclear envelope proteins known in early embryos. We used the yeast two-hybrid system to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA. Lamin's rod domain interacts with the complete otefin protein, with otefin's hydrophilic NH2-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA's COOH-terminal region is necessary and sufficient for interaction with lamin. Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina. They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.
Assuntos
Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Animais , Sítios de Ligação , Extratos Celulares , Núcleo Celular/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Laminas , Hibridização de Ácido Nucleico , Oócitos/metabolismo , Testes de Precipitina , Glândulas Salivares/metabolismo , Cloreto de SódioRESUMO
Otefin is a peripheral protein of the inner nuclear membrane in Drosophila melanogaster. Here we show that during nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. With the exception of sperm cells, otefin colocalizes with lamin Dm0 derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. In the egg chamber, otefin accumulates in the cytoplasm, in the nuclear periphery, and within the nucleoplasm of the oocyte, in a pattern similar to that of lamin Dm0 derivatives. There is a relatively large nonnuclear pool of otefin present from stages 6 to 7 of egg chamber maturation through 6 to 8 h of embryonic development at 25 degrees C. In this pool, otefin is peripherally associated with a fraction containing the membrane vesicles. This association is biochemically different from the association of otefin with the nuclear envelope. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. A major site for cdc2 kinase phosphorylation in vitro was mapped to serine 36 of otefin. Together, our data suggest an essential role for otefin in the assembly of the Drosophila nuclear envelope.
Assuntos
Cromatina/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/embriologia , Proteínas de Membrana/metabolismo , Membrana Nuclear/ultraestrutura , Proteínas Nucleares/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Compartimento Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Insetos/metabolismo , Laminas , Fosfoproteínas/metabolismo , Fosforilação , Fosfosserina/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and Ce-MAN1 migrate on SDS-PAGE as 17- and 52-kDa proteins, respectively. Based on their biochemical extraction properties and immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to determine the timing of nuclear envelope breakdown during mitosis in C. elegans. The C. elegans nuclear envelope disassembles very late compared with vertebrates and Drosophila. The nuclear membranes remained intact everywhere except near spindle poles during metaphase and early anaphase, fully disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos but existed until anaphase in 2- to 24-cell embryos. Intranuclear mRNA splicing factors disassembled after prophase. The timing of nuclear disassembly in C. elegans is novel and may reflect its evolutionary position between unicellular and more complex eukaryotes.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/genética , Ciclo Celular/fisiologia , Proteínas de Membrana/metabolismo , Membrana Nuclear/ultraestrutura , Poro Nuclear/fisiologia , Proteínas Nucleares/metabolismo , Timopoietinas/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/fisiologia , Proteínas de Ligação a DNA , Embrião não Mamífero/fisiologia , Humanos , Laminas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Metáfase , Mitose/fisiologia , Dados de Sequência Molecular , Poro Nuclear/ultraestrutura , Proteínas Nucleares/química , Proteínas Nucleares/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Timopoietinas/química , Timopoietinas/genéticaRESUMO
A Drosophila cell-free system was used to characterize proteins that are required for targeting vesicles to chromatin and for fusion of vesicles to form nuclear envelopes. Treatment of vesicles with 1 M NaCl abolished their ability to bind to chromatin. Binding of salt-treated vesicles to chromatin could be restored by adding the dialyzed salt extract. Lamin Dm is one of the peripheral proteins whose activity was required, since supplying interphase lamin isoforms Dm1, and Dm2 to the assembly extract restored binding. As opposed to the findings in Xenopus, okadaic acid had no effect on vesicle binding. Trypsin digestion of the salt-stripped vesicles eliminated their association with chromatin even in the presence of the dialyzed salt extract. One vesicles attached to chromatin surface, fusion events took place were found to be sensitive to guanosine 5'-[gamma-thio]triphosphate (GTP gamma S). These chromatin-attached vesicles contained lamin Dm and otefin but not gp210. Thus, these results show that in Drosophila there are two populations of nuclear vesicles. The population that interacts first with chromatin contains lamin and otefin and requires both peripheral and integral membrane proteins, whereas fusion of vesicles requires GTPase activity.
Assuntos
Cromatina/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Animais , Sistema Livre de Células , Cromatina/efeitos dos fármacos , Cromatina/ultraestrutura , Drosophila , Laminina/efeitos dos fármacos , Proteínas de Membrana/efeitos dos fármacos , Microscopia Eletrônica , Ácido Okadáico/farmacologiaRESUMO
Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs.
Assuntos
Caenorhabditis elegans/genética , Ciclo Celular/genética , Estruturas do Núcleo Celular/ultraestrutura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Estruturas do Núcleo Celular/metabolismo , Embrião não Mamífero , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/fisiologia , Laminas , Masculino , Membrana Nuclear/metabolismoRESUMO
CHox E is a novel chicken homeogene that belongs to the H2.0 family of homeodomains. Its homeobox sequence is interrupted by an intron between amino acids 44 and 45. Expression of CHox E during embryogenesis is localized to the central nervous system. The anterior boundary of CHox E expression can initially be localized to rhombomere number 1, later in development this boundary reaches up to the rhombencephalic isthmus. CHox E expression in the spinal cord localizes dorso-ventrally to the dorsal half of the basal plate. CHox E expression is always restricted to the proliferating region, the ventricular zone. As the ventricular zone becomes restricted laterally, so does the CHox E expressing region. Once this region of the ventricular zone ceases to exist, CHox E specific transcripts become undetectable. The site and time of CHox E expression suggest a very early function in the differentiation of the cells derived from that region of the ventricular zone.
Assuntos
Regulação da Expressão Gênica/fisiologia , Genes Homeobox/fisiologia , Medula Espinal/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Embrião de Galinha , DNA/genética , Desenvolvimento Embrionário e Fetal/fisiologia , Genes Homeobox/genética , Íntrons , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia , Medula Espinal/citologia , Medula Espinal/embriologia , Transcrição Gênica/genéticaRESUMO
We have isolated the novel murine Sax-1 gene, a member of the NK-1 class of homeobox genes, and report its expression pattern in the developing central nervous system (CNS) in comparison to two other homeobox genes, Evx-1 and Pax-6. Sax-1 was found to be transiently expressed in the developing posterior CNS. First seen in the ectoderm lateral to the primitive streak, the signal later encompassed the neural plate. Posteriorly, the expression domain overlapped with the Evx-1 expression in the streak, while anteriorly it was delimited by the Pax-6 signal in the neural tube. This early phase starting at day 9.5 pc, Sax-1 was expressed in distinct areas of spinal cord, hindbrain and forebrain. Particularly strong signals were detected in rhombomere 1 and in the pretectum. In these areas, subsets of neurons may be marked and specified. In addition to the normal pattern of Sax-1 during development, the expression in different mouse mutants was analysed. In Brachyury curtailed homozygotes, the expression of Sax-1 was found to be reduced during neurulation and even lost at day 9.0 pc. Ventral shift and finally loss of the signal in the ventral spinal cord was observed in Danforth's short tail homozygotes.
Assuntos
Sistema Nervoso Central/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Proteínas de Homeodomínio/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Proteínas Nucleares , Proteínas com Domínio T , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Sistema Nervoso Central/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas do Olho , Proteínas Fetais/genética , Gástrula/metabolismo , Idade Gestacional , Proteínas de Homeodomínio/genética , Hibridização In Situ , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Proteínas Repressoras , Rombencéfalo/metabolismo , Células Tumorais CultivadasRESUMO
The nuclear lamina is located between the inner nuclear membrane and the peripheral chromatin. It is composed mainly of nuclear lamins and lamina-associated proteins. The nuclear lamina is involved in nuclear organization, cell cycle regulation, and differentiation. As such, impairment in its architecture and/or function leads to genetic diseases and apoptosis. This article describes the molecular organization of the nuclear lamins, their assembly into filaments, their distribution within the nucleus, and the complex network of interactions between them and other proteins of the inner nuclear membrane. Recent findings unraveled evidence for specific interactions between proteins of the nuclear lamina and the chromatin. These include interactions between nuclear lamins and core histones, Lamina Associated Polypeptide 2 (LAP2), and the Barrier to Autointegration Factor (BAF) and interactions between lamin B receptor (LBR) and the chromodomain protein HP1. Taken together, these studies attribute a role for both the nuclear lamins and the lamina-associated proteins, LAP2 and LBR, in nuclear organization and nuclear assembly.
Assuntos
Núcleo Celular , Cromatina , Proteínas Nucleares , Animais , Núcleo Celular/química , Núcleo Celular/genética , Cromatina/química , Cromatina/genética , Humanos , Lamina Tipo B , Laminas , Proteínas Nucleares/química , Proteínas Nucleares/genéticaRESUMO
Activity of the cat gene driven by the cauliflower mosaic virus 35S promoter has been assayed by transfecting petunia protoplasts with the pUC8CaMVCAT plasmid. In vitro methylation of this plasmid with M.HpaII (methylates C in CCGG sites) and M.HhaI (methylates GCGC sites) did not affect bacterial chloramphenicol acetyltransferase (CAT) activity. It should be noted, however, that no HpaII or HhaI sites are present in the promoter sequence. In contrast, in vitro methylation of the plasmid with the spiroplasma methylase M.SssI, which methylates all CpG sites, resulted in complete inhibition of CAT activity. The promoter sequence contains 16 CpG sites and 13 CpNpG sites that are known to be methylation sites in plant DNA. In the light of this fact, and considering the results of the experiments presented here, we conclude that methylation at all CpG sites leaving CpNpG sites unmethylated is sufficient to block gene activity in a plant cell. Methylation of CpNpG sites in plant cells may, therefore, play a role other than gene silencing.
Assuntos
Regulação Enzimológica da Expressão Gênica , Protoplastos/metabolismo , Southern Blotting , Técnicas In Vitro , Metilação , Plasmídeos , TransfecçãoRESUMO
Several Drosophila genes involved in the control of segmentation and segment identity share a 183-bp conserved sequence termed homeo box. Homeo box sequences have been detected and cloned from the genomes of insects like Drosophila to vertebrates such as mouse and man. Two chicken homeo box genes CHox1 and CHox3, are described. Cloning of the CHox1 and CHox3 homeo boxes was performed using Drosophila and murine homeo box sequences as probes under low-stringency conditions. Analysis of both chicken homeo box sequences revealed them to be homeo boxes that have diverged from the Antennapedia class with homologies to homeo boxes of other organisms in the range of 75-42% at the nucleotide level and 69-41% at the protein level. Analysis of CHox3 expression during early embryo development showed that the gene codes for five transcripts 1.3, 1.9, 2.6, 5.6 and 7.9 kb in size. Three of the transcripts (1.3, 1.9 and 5.6 kb) are also recognized by a flanking non-homeo box containing probe. The levels of the different transcripts changed during the first five days of development. The most abundant transcripts (1.3 and 1.9 kb) are already present at the time the egg is laid. Their transcription peaks at day 1 of incubation and then decreases. The CHox1 transcripts are present at very low levels between days 2.5 and 4 of development. These two chicken genes represent bona fide Hox genes in a branch of vertebrates that evolved parallel to mammals.
Assuntos
Clonagem Molecular , Regulação da Expressão Gênica , Genes Homeobox , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , DNA/genética , Drosophila melanogaster/genética , Humanos , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Xenopus laevis/genéticaRESUMO
Chicken sperm chromatin initiated an assembly of interphase-like nuclei in a cell-free cytoplasmic preparation from 1-6 h old Drosophila melanogaster embryos. The formation of these interphase-like nuclei from the condensed sperm chromatin happened in a series of distinct steps. Anti-Drosophila lamin monoclonal antibody stained the assembled nuclei in a pattern indistinguishable from normal Drosophila nuclei. This assembly process required an ATP regenerating system and could be blocked by the addition of novobiocin into the cell-free extract.
Assuntos
Núcleo Celular/fisiologia , Cromatina/fisiologia , Membrana Nuclear/fisiologia , Espermatozoides/ultraestrutura , Trifosfato de Adenosina/fisiologia , Animais , Núcleo Celular/ultraestrutura , Sistema Livre de Células , Galinhas , DNA Topoisomerases Tipo II/fisiologia , Drosophila melanogaster , Técnicas In Vitro , Laminas , Masculino , Morfogênese , Novobiocina/farmacologia , Membrana Nuclear/ultraestrutura , Proteínas Nucleares/metabolismoRESUMO
Gbx2 homeobox genes are important for formation and function of the midbrain/hindbrain boundary, namely the isthmic organizer. Two Gbx2 genes were identified in Xenopus laevis, differing in 13 amino acids, including a change in the homeodomain. Xgbx2a is activated earlier during gastrulation and reaches higher levels of expression while Xgbx2b is expressed later, at lower levels and has an additional domain in the ventral blood islands. Their overexpression results in microcephalic embryos with shortened axes and defects in brain and notochord formation. Both genes encode functionally homologous proteins, which differ primarily in their temporal and spatial expression patterns.