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At least 793 records · Page 44Linked to original sources
PubMed2006

Histone acetylation-mediated chromatin compaction during mouse spermatogenesis.

One of the most dramatic chromatin remodelling events takes place during mammalian spermatogenesis involving massive incorporation of somatic and testis-specific histone variants, as well as generalized histone modifications before their replacement by new DNA packaging proteins. Our data suggest that the induced histone acetylation occurring after meiosis may direct the first steps of genome compaction. Indeed, a double bromodomain-containing protein expressed in postmeiotic cells, Brdt, shows the extraordinary capacity to specifically condense acetylated chromatin in vivo and in vitro. In elongating spermatids, Brdt widely co-localizes with acetylated histones before accumulating in condensed chromatin domains. These domains preferentially maintain their acetylation status until late spermatogenesis. Based on these data, we propose that Brdt mediates a general histone acetylation-induced chromatin compaction and also maintains differential acetylation of specific regions, and is therefore involved in organizing the spermatozoon's genome.

Acetylation↗
PubMed2006

Denaturing gradient gel electrophoresis can rapidly display the bacterial diversity contained in 16S rDNA clone libraries.

Two different strategies for molecular analysis of bacterial diversity, 16S rDNA cloning and denaturing gradient gel electrophoresis (DGGE), were combined into a single protocol that took advantage of the best attributes of each: the ability of cloning to package DNA sequence information and the ability of DGGE to display a community profile. In this combined protocol, polymerase chain reaction products from environmental DNA were cloned, and then DGGE was used to screen the clone libraries. Both individual clones and pools of randomly selected clones were analyzed by DGGE, and these migration patterns were compared to the conventional DGGE profile produced directly from environmental DNA. For two simple bacterial communities (biofilm from a humics-fed laboratory reactor and planktonic bacteria filtered from an urban freshwater pond), pools of 35-50 clones produced DGGE profiles that contained most of the bands visible in the conventional DGGE profiles, indicating that the clone pools were adequate for identifying the dominant genotypes. However, DGGE profiles of two different pools of 50 clones from a lawn soil clone library were distinctly different from each other and from the conventional DGGE profile, indicating that this small number of clones poorly represented the bacterial diversity in soil. Individual clones with the same apparent DGGE mobility as prominent bands in the humics reactor community profiles were sequenced from the clone plasmid DNA rather than from bands excised from the gel. Because a longer fragment was cloned (approximately 1500 bp) than was actually analyzed in DGGE (approximately 350 bp), far more sequence information was available using this approach that could have been recovered from an excised gel band. This clone/DGGE protocol permitted rapid analysis of the microbial diversity in the two moderately complex systems, but was limited in its ability to represent the diversity in the soil microbial community. Nonetheless, clone/DGGE is a promising strategy for fractionating diverse microbial communities into manageable subsets consisting of small pools of clones.

Bacteria↗
PubMed1998

The chromatin structure of the GAL1 promoter forms independently of Reb1p in Saccharomyces cerevisiae.

Positive and negative regulation of the GAL1 promoter of the yeast Saccharomyces cerevisiae results from a network of interactions between transcription factors and chromatin. In this study we used footprinting procedures to characterize these interactions in vivo. DNase I analysis of the GAL1 upstream activating sequence (UAS(GAL1/10)) showed expected Gal4 activator protein binding during growth in galactose, and also revealed binding of the Reb1 protein (Reb1p) during growth in glucose. In addition, we mapped to nucleotide resolution a positioned nucleosome that, in the inactive promoter, packages DNA between the UAS(GAL1/10) and the GAL1 TATA sequence, leaving both of these elements nucleosome free. The nucleosome footprint was lost when the promoter was activated. Surprisingly, mutation of the Reb1p binding site had no effect on nucleosome positioning or on the kinetics or extent of activation or repression of either the GAL1 or GAL10 promoters under any of the conditions assayed.

Base Sequence↗
PubMed1984

Bacteriophage lambda preconnectors. Purification and structure.

The morphogenesis of bacteriophage lambda proheads is under the control of the four phage genes B, C, Nu3 and E, and the two Escherichia coli genes groEL and groES . It has been shown previously that extracts prepared from cells infected with a lambda C-E- mutant accumulate a gpB polymer, which behaves as a biologically active intermediate in prohead assembly. This gpB activity has been called a preconnector , as it is probably a precursor to the head-tail connector. We now report the partial purification of biologically active preconnectors and the characterization of its structure. In the electron microscope, preconnectors appear as donut -like structures composed of several subunits displaying radial symmetry. Optical filtration of periodic arrays of preconnectors showed that the structure has 12-fold rotational symmetry. Side views of the preconnector reveal that it resembles an asymmetrical dumbell . This information has been used to construct a three-dimensional model of the preconnector . The implications of this structure for prohead shape and function, and for DNA packaging are discussed.

Bacteriophage lambda↗
PubMed1986

Three-dimensional reconstruction of the connector of bacteriophage phi 29 at 1.8 nm resolution.

The three-dimensional reconstruction of the connector of bacteriophage phi 29 has been obtained from tilt series of negatively stained tetragonal ordered aggregates under low-dose conditions and up to a resolution of (1/1.8) nm-1. These connectors are built up as dodecamers of only one structural polypeptide (p10). Two connectors form the crystal unit cell, each one facing in the opposite direction with respect to the plane of the crystal and partially overlapping. The main features of the two connectors that build the unit cell were essentially the same, although they were negatively stained in slightly different ways, probably due to their situations with respect to the carbon-coated support grid. The main features of the phi 29 connector structure revealed by this three-dimensional reconstruction are: the existence of two clearly defined domains, one with a diameter of around 14 nm and the other narrower (diameter approximately equal to 7.5 nm); an inner hole running all along the structure (around 7 to 8 nm in height) with a cylindrical profile and an average diameter of 4 nm; a general 6-fold symmetry along the whole structure and a 12-fold one in the wider domain; a clockwise twist of the more contrasted regions of both domains from the narrower towards the wider domain (the direction of DNA encapsidation). These features are compatible with an active role for the connector in the process of DNA packaging.

Bacteriophages↗
PubMed1991

Enteric adenovirus type 40: complementation of the E4 defect in Ad2 dl808.

The enteric adenovirus type 40 cannot be passaged in HeLa cells, but will grow productively in cells that express the E1B region of adenovirus types 2 or 5. Even in such permissive cells, the lytic cycle is prolonged, there is an abnormal pattern of E1B early gene expression and a failure to switch off host cell functions, suggesting that other gene functions might be impaired in Ad40. For Ad2, E4 ORF 6 and ORF 3 proteins are known to have an essential role in progressing from the early to the late phase of lytic infection and the shutoff of host functions requires an interaction between the E4 ORF 6 34K protein and the E1B 55K protein. To test whether E4 functions of Ad40 are impaired, complementation tests have been made between Ad40 and the E4 deletion mutant Ad2 dl808, which lacks all but ORF 1 of the E4 region. In HeLa and Vero cells, Ad40 complements dl808 to levels equivalent to an Ad2 wild-type infection, as demonstrated by measuring virion packaged DNA, virus titration, and viral protein synthesis. Surprisingly, Ad2 dl808 fails to reciprocally complement Ad40. The results show that Ad40 produces functional E4 ORF 6 and/or ORF 3 activity, and that their expression precedes DNA replication.

Adenovirus Early Proteins↗
PubMed1991

A pilot protein participates in the initiation of P22 procapsid assembly.

The gene 16 protein of the bacteriophage P22 is required as a pilot protein aiding the transfer of DNA from the phage into the Salmonella typhimurium host cell. During assembly 10-20 copies of the 63,000-Da gp 16 protein are incorporated into the procapsid shell prior to DNA packaging. The protein has been purified from isolated procapsids and behaved as a monomer in solution. Upon incubation with purified coat and scaffolding subunits in vitro, it assembled into procapsids with the correct stoichiometry. The addition of physiological quantities of gp 16 resulted in an increased rate of procapsid assembly. Sedimentation of mixtures of coat and gp 16 protein subunits revealed association/dissociation behavior. It is likely that the added gp 16 is acting to stabilize a transient oligomeric coat protein species that functions as the in vitro initiation complex for procapsid assembly.

Capsid↗
PubMed1995

Temperature induction of bacteriophage lambda mutants in Escherichia coli.

The paper presents temperature induction in Escherichia coli cells with phage lambda on the target-protein production and cell growth. Replicated lambda-DNA particles in the Q- and S- mutants remain naked for a longer time by preventing DNA packaging and cell lysis, and therefore the expression of the foreign genes is high. However, the parasitic infection of phage-lambda causes on significant losses of host cell viability in the induction phase. The temperature effects on cell growth and targeted-gene product formation were investigated. Gene amplification was found to be growth phase dependent for both Qam73 (Q mutation) and Sam100 (S mutation) mutants. Maximum induction occurs in the early exponential phase and under the optimal cell density. The total beta-galactosidase activity at this optimal induction condition increases roughly 8-10-fold with respect to that without thermal induction. To maximize the induction efficiency for the gene-product beta-galactosidase activity, several operating parameters were investigated. In this study, temperature induction is strongly dependent upon the population density of 'susceptible' cells at which time the temperature is shifted to 38-42 degrees C. This may be due to the 'threshold' population density to regulate the infection of lambda to hosts and control the productivity of target gene expression.

Bacteriophage lambda↗
PubMed1984

Carcinogenesis--devolution towards an ancient nucleated pre-eukaryotic level.

Because the mitochondria and the cells housing them are obligatory symbionts, the evolutionary history of cells forms the locus minoris resistentiae which is the prerequisite for the carcinogenetic process. During carcinogenesis, the cells devolve towards an ancient anaerobic nucleated pre-eukaryotic level. True carcinogens cause an accumulation of inclusion bodies in the inner, bacterial, mitochondrial membrane. The mitochondrial damage which is detectable only in the early pretumorous stages, results in the respiratory surface with its enzymes being specifically changed, the mitochondrial and nuclear cycles no longer coinciding, the energy generation being forced to reuse the latent, "prehistoric", mode of respiration and the mitochondrial enzyme systems of soil bacterial origin becoming adapted to use other and more versatile metabolic pathways with a wider variety of end-products than classical glycolysis which produces lactate only. Neither external carcinogens nor oncogens are necessary. An increased, prolonged cell replication activity of physiological type is sufficient to initiate and maintain the process in animals with an inherited neoplastic disposition located in the inner mitochondrial membrane. The neoplastic disposition is inherited maternally: in fertilization the ovum does not receive mitochondria from the spermatocyte. The final results are an overall retardation of cell processes and instability in its structural and functional repertoire, the cytoskeleton (differentiation organelle) of the malignant cell manifesting special patterns. The proposed devolutionary mechanism is feasible as DNA packages are physiological components of soil bacterial membrane and can remain dormant (repressed) for years, or for ever, but under suitable conditions can generate seemingly new species, and particularly because enzyme adaptability is the unique privilege of soil bacteria.

Animals↗
PubMed1995

Telomere structure and function.

Telomeres, the termini of linear eukaryotic chromosomes, contain specific DNA sequences that are widely conserved. These sequences not only recruit telomere-specific proteins, but also give telomeric DNA the ability to fold into four-stranded DNA structures. Recent structural studies have shown that the repertoire of quadruplexes formed by the G-rich strand is larger than had been envisaged. Even more surprising is a novel four-stranded structure formed by the C-rich strand, called the i-tetraplex. Genetic and biochemical analyses have been used to identify proteins involved in telomeric DNA packaging and organization. The possibility that four-stranded structures may play a role in telomere function has been strengthened by the discovery that telomeric proteins can bind to and promote the formation of G-quadruplexes.

Base Sequence↗
PubMed2006

The analysis of histone modifications.

The biological function of many proteins is often regulated through posttranslational modifications (PTMs). Frequently different modifications influence each other and lead to an intricate network of interdependent modification patterns that affect protein-protein interactions, enzymatic activities and sub-cellular localizations. One of the best-studied class of proteins that is affected by PTMs and combinations thereof are the histone molecules. Histones are very abundant, small basic proteins that package DNA in the eukaryotic nucleus to form chromatin. The four core-histones are densely modified within their first 20-40 N-terminal amino acids, which are highly evolutionary conserved despite playing no structural role. The modifications are thought to constitute a histone code that is used by the cell to encrypt various chromatin conformations and gene expression states. The analysis of modified histones can be used as a model to dissect complex modification patterns and to investigate their molecular functions. Here we review techniques that have been used to decipher complex histone modification patterns and discuss the implication of these findings for chromatin structure and function.

Amino Acid Sequence↗
PubMed2004

The novel histone deacetylase inhibitor BL1521 inhibits proliferation and induces apoptosis in neuroblastoma cells.

Neuroblastoma is a childhood cancer arising from the sympathetic nervous system. Disseminated neuroblastoma has a poor prognosis despite intensive multimodality treatment. Histone deacetylases (HDACs) were recently discovered as a potential target for pharmacological gene therapy in cancer. HDACs have an important function in regulating DNA packaging in chromatin, thereby affecting the transcription of genes. In this paper, we tested the efficacy of a newly developed histone deacetylase inhibitor, BL1521, on neuroblastoma in vitro by investigating the changes in: acetylation of histone H3, in situ HDAC activity, p21(WAF1/CIP1) and MYCN expression, metabolic activity, proliferation, morphology and the amount of apoptosis present. BL1521 inhibited the in situ HDAC activity of a panel of neuroblastoma cell lines by at least 85%. Western analysis showed an increase of histone H3 acetylation in neuroblastoma cells after incubation with BL1521. Northern analysis showed an increase in the expression of p21(WAF1/CIP1) and a decrease in the expression of MYCN in neuroblastoma cells after incubation with BL1521. Proliferation as well as the metabolic activity of neuroblastoma cells decreased significantly in response to treatment with BL1521, regardless of the MYCN status of the cells. BL1521 induced poly-(ADP-ribose) polymerase cleavage in a time- and dose-dependent manner, indicating the induction of apoptosis. Furthermore, when compared to the HDAC inhibitors Trichostatin A and 4-phenylbutyrate, BL1521 has an intermediate efficacy. Our results show that BL1521 is a potent inhibitor of HDAC and that HDACs are an attractive target for selective chemotherapy in neuroblastoma.

Antineoplastic Agents↗
PubMed2005

Domain study of bacteriophage p22 coat protein and characterization of the capsid lattice transformation by hydrogen/deuterium exchange.

Viral capsids are dynamic structures which undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Limited proteolysis and biochemical experiments indicated that the coat protein consists of two domains connected by a flexible loop. To investigate the properties and roles of the sub-domains, we have cloned them and initiated structure and function studies. The N-terminal domain, which is made up of 190 amino acid residues, is largely unstructured in solution, while the C-terminal domain, which consists of 239 amino acid residues, forms a stable non-covalent dimer. The N-terminal domain adopts additional structure in the context of the C-terminal domain which might form a platform on which the N-terminal domain can fold. The local dynamics of the coat protein in both procapsids and mature capsids was monitored by hydrogen/deuterium exchange combined with mass spectrometry. The exchange rate for C-terminal domain peptides was similar in both forms. However, the N-terminal domain was more flexible in the empty procapsid shells than in the mature capsids. The flexibility of the N-terminal domain observed in the solution persisted into the procapsid form, but was lost upon maturation. The loop region connecting the two domains exchanged rapidly in the empty procapsid shells, but more slowly in the mature capsids. The global stabilization of the N-terminal domain and the flexibility encoded in the loop region may be a key component of the maturation process.

Amino Acid Sequence↗
PubMed2006

Shape and compaction of Escherichia coli nucleoids.

The genomic DNA in cells of Escherichia coli is localized in one or two compact, phase-like regions with characteristic shapes. Nucleoids undergo progressive changes in shape and compaction in the presence of drugs such as chloramphenicol or puromycin. Forces which influence nucleoid shape and compaction are reviewed, with particular emphasis on crowding effects of the cytoplasm and confinement effects of the cell envelope. Based in part on the theory of Kornyshev and Leikin for interaction between DNA duplexes, the folding of DNA caused by binding of DNA-associated proteins is suggested to antagonize DNA condensation and, thereby, increase access to DNA sequences. These views are incorporated into a working model for nucleoid organization.

Chromosomes, Bacterial↗
PubMed2006

Structural and physical aspects of bacterial chromosome segregation.

Microscopic observations on the bacterial nucleoid suggest that the chromosome occurs in the cell as a compact nucleoid phase separate from the cytoplasm. Physical theory likewise predicts a phase separation, taking into consideration DNA supercoiling, nucleoid-binding proteins, and excluded-volume interactions between DNA and cytoplasmic proteins. Specific DNA loci, visualized as oriC-GFP spots in the densely packed nucleoid, exhibit a very low diffusion coefficient indicating that they are virtually immobile and may primarily be moved by overall length growth. Such gradual movement could be effectuated by replication, transertion (combined transcription, translation, and insertion of proteins), and actin- (MreB) directed surface synthesis. Differences in the movement and positioning of gene loci between Escherichia coli and Caulobacter crescentus are discussed. We propose that a low diffusion coefficient could explain the linear positioning of genes in the nucleoid and that differential transcriptional activity could induce different mobilities between either replichores (E. coli) or daughter strands (C. crescentus). The transertion process, possibly in combination with MreB cytoskeletal tracks, could overcome the compaction forces and move specific chromosomal regions and the nucleoid as a whole without invoking a dedicated mechanism.

Chromosome Segregation↗
PubMed2006

The architectural role of nucleoid-associated proteins in the organization of bacterial chromatin: a molecular perspective.

The bacterial genome is folded into a compact structure called the nucleoid. Considerable compaction of the DNA molecule is required in order to reduce its volume below that of the cell. Several mechanisms, such as molecular crowding and DNA supercoiling contribute to the compactness of the nucleoid. Besides these mechanisms, a number of architectural proteins associate with the chromosomal DNA and cause it to fold into a compact structure by bridging, bending or wrapping DNA. In this review, we provide an overview of the major nucleoid-associated proteins from a structural perspective and we discuss their possible roles in dynamically shaping the bacterial nucleoid.

Bacterial Proteins↗
PubMed2006

Chromosome organization and segregation in bacteria.

In the recent years, considerable advances have been made towards understanding the structure and function of the bacterial chromosome. A number of different factors appear to cooperate in condensing DNA into a highly dynamic assembly of supercoiled loops. Despite this variability in the lower levels of chromatin structure, the global arrangement of chromosomal DNA within the cell is surprisingly conserved, with loci being arrayed along the cellular long axis in line with their order on the genomic map. This conserved pattern is propagated during the course of DNA segregation. First, after entry into S-phase, the newly synthesized origin regions are segregated in an active and directed process, involving the bacterial actin homolog MreB. Subsequent DNA segments then follow by different mechanisms. They are separated immediately after release from the replisome and move rapidly to their conserved positions in the incipient daughter cell compartments. Partitioning of the bacterial chromosome thus takes place while DNA replication is in progress.

Bacteria↗
PubMed2004

Chromatin remodeling by RNA polymerases.

Chromatin packages DNA tightly into the eukaryotic nucleus and maintains its proper functioning. Recent studies suggest the existence of two distinct mechanisms of progression of RNA polymerases through chromatin. The first is characteristic of eukaryotic RNA polymerase III, bacteriophage RNA polymerases, and probably ATP-dependent chromatin remodeling complexes. In this mechanism, nucleosomes are translocated without release of the octamer into solution. By contrast, transcription by RNA polymerase II (Pol II) involves displacement of one H2A-H2B dimer. Nucleosomes can present a barrier for transcribing Pol II that can be regulated in vivo. Analysis of the mechanisms of transcription through chromatin should provide important information about mechanisms of chromatin remodeling and gene regulation at the level of transcript elongation.

Adenosine Triphosphate↗