RESUMO
Transgenic clones of mouse embryonic stem cells of the RI line were -received by transfection of plasmid linear vectors. The changes in the transgene structure during its integration into the genome of the target cells were investigated. Displacements were found on the flanks of the integrated transgene. It was found that multicopy tandem structures are formed in head- -tail orientation at the transgene integration. It was noted that the number of copies of the integrated transgenes varies considerably, but the introduction of DNA fragments from the nuclear shells into the flanks of the transgene normalizes the number of its copies.
Assuntos
Dosagem de Genes , Células-Tronco Embrionárias Murinas/metabolismo , Transfecção , Transgenes , Animais , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologiaRESUMO
The results of investigating the functions of different barrier elements, including insulators and MARs/SARs and the models of their effect, are described. The functions of neDNA (DNA from the nuclear envelopes) as a barrier element that protects the transgene from position-effect variegation and its interaction with insulators are discussed. The possible mechanisms of the functioning of structural and functional units of eukaryotic chromosomes of different species are suggested.
Assuntos
Cromatina/química , Elementos Isolantes , Membrana Nuclear/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , Efeitos da Posição Cromossômica , Humanos , Proteínas Associadas à Matriz Nuclear/metabolismo , TransgenesRESUMO
A neDNA fragment that protects a transgene from position effect variegation when flanking it was tested for insulator properties. The fragment did not act as an insulator. A similarity of neDNA and various barrier elements was examined, and Drosophila melanogaster and Mus musculus chromosome DNA regions homologous to neDNA were analyzed. Additional conserved sites and DNA duplex destabilization sites were found in the neDNA sequence, and DNA conformational specifics were assumed for the chromosomal neighborhood of neDNA sites of the D. melanogaster and M. musculus genomes.
Assuntos
Cromossomos/genética , DNA/genética , Drosophila melanogaster/genética , Membrana Nuclear/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases/genética , Efeitos da Posição Cromossômica/genética , Sequência Conservada/genética , Regulação da Expressão Gênica , Elementos Isolantes/genética , Interfase/genética , Camundongos , Conformação de Ácido NucleicoRESUMO
To study possible involvement of polypurine and polypyrimidine DNA tracks potentially capable to form triple-stranded structures (H-form of DNA) in compactization of eukaryotic chromosomes a search in silico for "complementary" polypurine and polypyrimidine tracks within 12 eukaryotic gene nucleotide sequences was carried out. Polypurine and polypyrimidine tracks (10-11 b.p.) potentially capable of interacting with each other with the formation of triplex structures ("structurizing" regions) has been shown to be located in chromosomal locus of genes, predominantly in introns and flanking regions. In the case of in vivo realization of such DNA-DNA interactions the chromosomal gene domains can be folded into several small loops. An involvement of DNA triplexes in the chromosomal gene loci compactization may be associated with the gene functioning. The analogous analysis carried out for nucleotide sequences of long (LINE) and short (SINE) repeats dispersed over the genome as well as of satellite DNA has demonstrated a fundamental identity between mechanisms of chromosomal encoding and non-coding regions' compactization.
Assuntos
Cromatina/química , Cromossomos de Mamíferos/química , DNA/química , Conformação de Ácido Nucleico , Animais , Cromatina/genética , Cromossomos de Mamíferos/genética , Biologia Computacional , DNA/genética , DNA Satélite/química , DNA Satélite/genética , Humanos , Íntrons/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Camundongos , Coelhos , RatosRESUMO
The functional characteristics of the DNA fragments responsible for chromosome attachment to the nuclear envelope during the interphase (neDNAs) have been studied. The neDNAs flanking the transgene have been found to promote a steadily high rate of its expression, irrespective of the site of its insertion into the host chromosomes. At the same time, neDNAs themselves have no transcription regulatory functions.
Assuntos
Cromossomos/fisiologia , Membrana Nuclear/fisiologia , Proteínas Nucleares/genética , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Animais Geneticamente Modificados , DNA/fisiologia , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster , Cor de Olho/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica , Genes Reporter , Interfase , Membrana Nuclear/genética , TransgenesRESUMO
The survival of transgenic mouse embryos was studied as a function of the transgene structure. The data obtained indicate that the introduction of a chromosomal DNA fragment providing for the anchoring of interphase chromosomes on the nuclear envelope increases the efficiency of transgenesis in mice threefold due to their increased viability.
Assuntos
Cromossomos de Mamíferos/genética , Embrião de Mamíferos/metabolismo , Técnicas de Transferência de Genes , Interfase/fisiologia , Membrana Nuclear/metabolismo , Transgenes/fisiologia , Animais , Cromossomos de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Membrana Nuclear/genéticaRESUMO
Fragments of chromosomal DNA isolated from nuclear envelopes of mouse hepatocytes were previously cloned and partially characterized in our laboratory. One of the cloned fragments (EnvM4) had unusual characteristics: abundant representability in the clone library (about 50%) and homology with DNA of archebacteria. An analysis of nucleotide sequence of this fragment conducted in more detail allowed the detection of an evolutionary conserved region present in the genomes of various organisms (from bacteria to human). Other characteristics of fragment EnvM4 suggest a possible functional role of chromosomal DNA regions determining the attachment of chromosomes to the nuclear envelope.
Assuntos
DNA/genética , Evolução Molecular , Hepatócitos/química , Membrana Nuclear/química , Animais , Sequência de Bases , Sequência Conservada , DNA/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
We analyzed the possibility of triplex formation in the human alpha 1-antitrypsin gene. Conditions and nucleotide sequence dependence of triplex formation and thermostability of the product were determined.
Assuntos
DNA/química , alfa 1-Antitripsina/genética , Humanos , Conformação de Ácido Nucleico , Oligonucleotídeos/química , TemperaturaRESUMO
Scaffold/matrix-associated region (S/MAR) sequences are DNA regions that are attached to the nuclear matrix, and participate in many cellular processes. The nuclear matrix is a complex structure consisting of various elements. In this paper we compared frequencies of simple nucleotide motifs in S/MAR sequences and in sequences extracted directly from various nuclear matrix elements, such as nuclear lamina, cores of rosette-like structures, synaptonemal complex. Multivariate linear discriminant analysis revealed significant differences between these sequences. Based on this result we have developed a program, ChrClass (Win/NT version, ftp.bionet.nsc.ru/pub/biology/chrclass/chrclass.zip), for the prediction of the regions associated with various elements of the nuclear matrix in a query sequence. Subsequently, several test samples were analyzed by using two S/MAR prediction programs (a ChrClass and MAR-Finder) and a simple MRS criterion (S/MAR recognition signature) indicating the presence of S/MARs. Some overlap between the predictions of all MAR prediction tools has been found. Simultaneous use of the ChrClass, MRS criterion and MAR-Finder programs may help to obtain a more clearcut picture of S/MAR distribution in a query sequence. In general, our results suggest that the proportion of missed S/MARs is lower for ChrClass, whereas the proportion of wrong S/MARs is lower for MAR-Finder and MRS.
Assuntos
DNA/genética , Matriz Nuclear/genética , Animais , Sequência de Bases , DNA/isolamento & purificação , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Grão Comestível/genética , Globinas/genética , Humanos , Dados de Sequência Molecular , Matriz Nuclear/química , Oryza/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/estatística & dados numéricos , Software , Telômero/genéticaAssuntos
Simulação por Computador , DNA/genética , Matriz Nuclear/genética , Animais , Antígenos Nucleares , Sítios de Ligação/genética , DNA/metabolismo , Humanos , Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Valor Preditivo dos Testes , Ligação Proteica , Análise de Sequência de DNARESUMO
Attachment regions of the eukaryotic chromosomal DNA to the nuclear scaffold/matrix (S/MARs) participate in various important cellular processes. However, no obvious characteristics common for these nucleotide sequences have been revealed, except that S/MARs are non-coding sites containing putative regulatory elements and binding sites of DNA-topoisomerase II. Heterogeneity among S/MARs can be caused by a variety of biological factors. In this paper, the accuracy of two S/MARs prediction programs, MAR-Finder (Singh, Kramer and Krawetz, 1997) and ChrClass (Glazkov, Rogozin and Glazko, 1998) are compared and it is concluded that both programs can be recommended for analysis of eukaryotic genomes. However, results of their prediction should be interpreted with caution since estimation of prediction accuracy of both programs needs further analysis. Problems of S/MARs prediction are illustrated on several examples of human protein-coding genes, repeated elements and the beta-globin locus from different mammalian species. Results of our analysis suggest that the proportion of missed S/MARs is lower for ChrClass, whereas the proportion of wrong S/MARs is lower for MAR-Finder (a default set of parameters).
Assuntos
Biologia Computacional , Genes , Matriz Nuclear/genética , Proteínas/genética , Animais , Biologia Computacional/métodos , DNA/química , DNA/genética , Globinas/genética , Humanos , Mamíferos , Matriz Nuclear/química , Proteínas/química , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Reprodutibilidade dos Testes , Análise de Sequência de DNA/métodos , SoftwareAssuntos
DNA , Células Eucarióticas , Modelos Genéticos , Conformação de Ácido Nucleico , Animais , HumanosRESUMO
Six DNA fragments of interphase chromosomes isolated from nuclear envelopes of murine hepatocytes were cloned and sequenced. Analysis of their structural-functional organization suggests that these fragments are highly specified protein-nonencoding fractions of a eukaryotic genome. In the evolutionary process, they appear already in archaebacteria and may be "ancestral" for DNA sequences involved in structuring chromosomal domains (rosette-like structures) of tissue-specific genes. In their composition, these fragments have nucleotide sequences homologous to the repeats of the SINE and LINE families and to the satellite DNA of murine centromeres.
Assuntos
Cromossomos/genética , DNA/genética , Membrana Nuclear/genética , Análise de Sequência de DNA , Animais , Sequência de Bases , Cromossomos/ultraestrutura , Interfase , Camundongos , Dados de Sequência Molecular , Membrana Nuclear/ultraestruturaRESUMO
The role of chromosome-membrane associations in order of spatial chromosome organization in interphase nuclear is reviewed. Data on specificity and mechanism of interphase chromosome anchoring on nuclear membrane are summarized.
Assuntos
Núcleo Celular/ultraestrutura , Cromossomos Humanos/ultraestrutura , Cromossomos/ultraestrutura , Interfase/genética , Membranas Intracelulares/ultraestrutura , Animais , Núcleo Celular/genética , Cromossomos/genética , Cromossomos Humanos/genética , DNA/genética , DNA/ultraestrutura , HumanosRESUMO
The problem of structural and functional units of eukaryotic chromosomes is considered. The results of our studies of elementary chromomeres having the rosettelike organization are summarized. According to our data, the rosettelike structures are the universal chromosomal substructures of animal somatic and meiotic cells. Chromosomal domains of transcriptionally inactive genes are organized as the rosettelike structures. The mechanism underlying the formation of rosettelike chromatin structures is discussed, including the specific DNA-protein interaction and the participation of noncanonical DNA forms (H-form).
Assuntos
Cromossomos/ultraestrutura , Células Eucarióticas/ultraestrutura , AnimaisRESUMO
Eukaryotic DNA topoisomerase II was shown to catalyze recombination between circular supercoiled plasmid DNAs in vitro. The results obtained are discussed with regard to the organization of eukaryotic chromosomes in the form of topologically independent domains (loops).
Assuntos
DNA Topoisomerases Tipo II/genética , DNA Circular/genética , DNA Super-Helicoidal/genética , Escherichia coli/genética , Plasmídeos/genética , Recombinação Genética , Catálise , Escherichia coli/enzimologiaRESUMO
Extensive evidence indicates that eukaryotic chromosomes are organized as sets of structural-functional units (domains) that form the basis of independent gene activity. At present, several types of chromosome regions have been suggested for the role of putative domain boundaries between structural-functional units in eukaryotic chromosomes. In this review, the experimental data on several chromosome elements as possible boundaries in eukaryotic chromosomes are presented. They are: scs/scs'-elements of the Drosophila heat-shock locus, DCR, LCR and the insulator of the human and chicken beta-globin locus, as well as MARs (SARs) of plant and animal chromosomes.
