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PubMed1999

Characterization of thermostable RecA protein and analysis of its interaction with single-stranded DNA.

Thermostable RecA protein (ttRecA) from Thermus thermophilus HB8 showed strand exchange activity at 65 degrees C but not at 37 degrees C, although nucleoprotein complex was observed at both temperatures. ttRecA showed single-stranded DNA (ssDNA)-dependent ATPase activity, and its activity was maximal at 65 degrees C. The kinetic parameters, K(m) and kcat, for adenosine triphosphate (ATP) hydrolysis with poly(dT) were 1.4 mM and 0.60 s-1 at 65 degrees C, and 0.34 mM and 0.28 s-1 at 37 degrees C, respectively. Substrate cooperativity was observed at both temperatures, and the Hill coefficient was about 2. At 65 degrees C, all tested ssDNAs were able to stimulate the ATPase activity. The order of ATPase stimulation was: poly(dC) > poly(dT) > M13 ssDNA > poly(dA). Double-stranded DNAs (dsDNA), poly(dT).poly(dA) and M13 dsDNA, were unable to activate the enzyme at 65 degrees C. At 37 degrees C, however, not only dsDNAs but also poly(dA) and M13 ssDNA showed poor stimulating ability. At 25 degrees C, poly(dA) and M13 ssDNA gave circular dichroism (CD) peaks at around 192 nm, which reflect a particular structure of DNA. The conformation was changed by an upshift of temperature or binding to Escherichia coli RecA protein (ecRecA), but not to ttRecA. The dissociation constant between ecRecA and poly(dA) was estimated to be 44 microM at 25 degrees C by the change in the CD. These observations suggest that the capability to modify the conformation of ssDNA may be different between ttRecA and ecRecA. The specific structure of ssDNA was altered by heat or binding of ecRecA. After this alteration, ttRecA and ecRecA can express their activities at each physiological temperature.

Adenosine Triphosphatases↗

PubMed2001

High local protein concentrations at promoters: strategies in prokaryotic and eukaryotic cells.

The speed of chemical reactions is proportional to the concentration of molecules involved. Since proteins catalyze most of the essential reactions inside a living cell, their concentration should be as high as possible. An economical way to achieve this is through the establishment of small cell compartments. We propose that within these compartments, two types of local concentration effects are at work. (1) With local concentration type I reactions, multimeric proteins bound to a specific DNA sequence have an increased local concentration for a second DNA site sufficiently close-by, or for proteins bound to such a site. (2) For type II effects, DNA can be used as a scaffold to build unique nucleoprotein complexes that would otherwise not exist free in solution. These complexes are proficient in establishing longer-range interactions with similarly unique complexes located far away on the genome. We discuss the consequences of these local concentration effects in the light of the markedly different sizes of prokaryotic and eukaryotic cells and of their genomes.

Animals↗

PubMed2004

Dynamic regulation of DNA methylation coupled transcriptional repression: BDNF regulation by MeCP2.

A recurrent theme in eukaryotic genome regulation stipulates that the properties of DNA are strongly influenced by the nucleoprotein complex into which it is assembled. Methylation of cytosine residues in vertebrate genomes has been implicated in influencing the assembly of locally repressive chromatin architecture. Current models suggest that covalent modification of DNA results in heritable, long-term transcriptional silencing. In October of 2003, two manuscripts were published that challenge important aspects of this model, suggesting that modulation of both DNA methylation itself, as well as the machinery implicated in its interpretation, are involved in acute gene regulation.

Animals↗

PubMed1984

Nude mice injected with DNA released by antigen stimulated human T lymphocytes produce specific antibodies expressing human characteristics.

Nude mice were injected with DNA purified from the nucleoprotein complex released by T lymphocytes previously exposed in vitro to inactivated herpes or poliovirus. After five days the serum of these mice was tested for its virus neutralizing activity. Results show that injected nude mice synthesize antiherpetic or antipolio antibodies depending on the antigen used to sensitize the T lymphocytes in vitro. These antibodies were not found in the serum of uninjected control mice or mice injected with inactivated herpes or polio viruses. Mice injected with DNA released by human T cells produced antibodies carrying human allotypes since they could be neutralized by anti-allotype sera. Moreover their antiviral activity was inhibited by anti-human IgM or IgG. However, the mice which were injected with DNA released by antigen stimulated murine T lymphocytes produced antiviral antibodies which were not neutralized by anti-human allotype sera.

Animals↗

PubMed2002

Enhanced expression of telomerase activity in thymoma and thymic carcinoma tissues: a clinicopathologic study.

BACKGROUND: Telomerase is a nucleoprotein complex that caps the physical termini of all eukaryotic chromosomes. Because most malignant cells and reproductive cells have telomerase activity, which elongates telomeric DNA, telomerase may play important roles in unlimited cell division acquisition of the malignant phenotype. The current study examined the relation of telomerase activity in thymoma and thymic carcinoma with the clinicopathologic features of these lesions. METHODS: Tissue specimens were surgically resected from patients with thymoma and thymic carcinoma. Telomerase activity was evaluated according to a modified telomeric repeat amplification protocol assay. Paraffin sections of tumor were immunostained by MIC2 antibody, a marker of immature T cells. RESULTS: Telomerase activity was detected in all thymic epithelial tumors. The activity (mean +/- SD; unit per microg protein) in thymoma (n = 17) was significantly higher than that in thymic carcinoma (n = 7) (431.8 +/- 400.1 vs. 68.8 +/- 39.8; P < 0.01). Telomerase activities in thymoma and thymic carcinoma were significantly higher than that in primary lung adenocarcinoma (33.5 +/- 39.2, n = 47), studied as a control (P < 0.01). In patients with thymoma, telomerase activity did not correlate with tumor stage according to Masaoka classification (P = 0.776). In patients with thymic carcinoma, however, telomerase activity positively correlated with tumor stage (P = 0.02). In thymoma, telomerase activity positively correlated with the ratio of induced lymphocytes according to Rosai's classification (P = 0.045). MIC2-positive lymphocytes were identified in all cases of thymoma (n = 12). In contrast, lymphocytes infiltrating thymic carcinoma did not react with MIC2. CONCLUSIONS: In thymoma, telomerase activity reflects the presence of immature T-cell lymphocytes in tumor tissue rather than tumor stage or malignant phenotype. In thymic carcinoma, telomerase activity derived directly from cancer cells may relate to tumor stage.

12E7 Antigen↗

PubMed2006

Critically short telomeres in acute myeloid leukemia with loss or gain of parts of chromosomes.

Telomeres, nucleoprotein complexes at chromosome ends, protect chromosomes against end-to-end fusion. Previous in vitro studies in human fibroblast models indicated that telomere dysfunction results in chromosome instability. Loss of telomere function can result either from critical shortening of telomeric DNA or from loss of distinct telomere-capping proteins. It is less clear whether telomere dysfunction has an important role in human cancer development in vivo. Acute myeloid leukemia (AML) is a good model to study mechanisms that generate chromosome instability in human cancer development because distinct groups of AML are characterized either by aberrations that theoretically could result from telomere dysfunction (terminal deletions, gains/losses of chromosome parts, nonreciprocal translocations), or aberrations that are unlikely to result from telomere dysfunction (e.g., reciprocal translocations or inversions). Here we demonstrate that AML with multiple chromosome aberrations that theoretically could result from telomere dysfunction is invariably characterized by critically short telomeres. Short telomeres in this group are not associated with low telomerase activity or decreased expression of essential telomeric capping proteins TRF2 and POT1. In contrast, telomerase activity levels are significantly higher in AML with short telomeres. Notably, short telomeres in the presence of high telomerase may relate to significantly higher expression of TRF1, a negative regulator of telomere length. Our observations suggest that, consistent with previous in vitro fibroblast models, age-related critical telomere shortening may have a role in generating chromosome instability in human AML development.

Acute Disease↗

PubMed1983

Electron microscopic studies of the different topological forms of the cauliflower mosaic virus DNA: knotted encapsidated DNA and nuclear minichromosome.

Cauliflower mosaic virus (CaMV) DNA exists under different topological forms in infected plants. First, the population of encapsidated CaMV DNA molecules appears heterogeneous when analysed by gel electrophoresis. The electron microscopic study reported here reveals that CaMV virion DNA contains simple and multiple topological knots. Second, a supercoiled DNA form never found in virions exists as a chromatin-like nucleoprotein complex with nucleosome subunits in the nuclei of infected leaves. The compaction ratio of the minichromosomes is compatible with the nucleosomal structure, the number of nucleosomes (41.0 +/- 2.5) is in keeping with the length of the viral genome.

Cell Nucleus↗

PubMed1986

Electron microscopic study of DNA complexes with proteins from the Archaebacterium Sulfolobus acidocaldarius.

DNA-protein complexes formed in vitro with isolated DNA-binding proteins from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius were analyzed by electron microscopy. Two of the proteins (10a and 10b) formed specific structures after incubation with DNA. Protein 10a, at low protein concentrations, showed individual small spots on the DNA and at high concentrations evenly covered doublestranded DNA. Protein 10b showed three different types of regular structures: one with single-stranded and two with double-stranded DNA. Using double-stranded DNA, 10b first bound cooperatively to two strands forming long, plait-like structures only slightly shorter than respective free DNA. The complex consists of two right-handed, interwound fibers, with a helical pitch of 26 nm and a diameter of approximately 10-11 nm. At higher protein concentration than needed to package all DNA into the complex with two double-stranded DNAs, the two DNAs were separated again and a new structure was formed evenly covering only one DNA strand. Both structures showed no significant contraction of the length of the DNA covered in the complex. Nucleoprotein formed with single-stranded PhiX174 DNA had a diameter of approximately 11 nm and could form circles with a contour length of 0.5 mum.

Journal Article↗

PubMed1988

Resolution of ColE1 dimers requires a DNA sequence implicated in the three-dimensional organization of the cer site.

Plasmid ColE1 specifies a recombination site (cer) which participates in the conversion of plasmid dimers to monomers. The uncontrolled accumulation of dimers (and higher oligomeric forms) would otherwise lead to plasmid instability. Exonuclease III-generated deletions have been used to define the left-hand boundary of the cer site. Deletions which have lost up to 60 bp adjacent to the boundary no longer mediate the conversion of plasmid dimers to monomers, but still recombine with a wild-type site. Although this boundary region is essential for dimer resolution, its DNA sequence is poorly conserved among multimer resolution sites in related plasmids. We present evidence that its function is to influence the three-dimensional organization of the site and suggest that it may be required for the formation of a condensed nucleoprotein complex.

Chromosome Deletion↗

PubMed1989

Co-operative interactions between NFI and the adenovirus DNA binding protein at the adenovirus origin of replication.

The DNA-protein and protein-protein interactions proposed for the stability of nucleoprotein complexes at the origin of replication in prokaryotes are also thought to impart regulatory precision in eukaryotic DNA replication. This type of specificity can be observed, for example, during adenovirus DNA replication where efficient initiation requires that nuclear factor I (NFI) binds to the origin of DNA replication. Addition of purified NFI stimulates the initiation of adenovirus DNA replication in vitro in a reaction that is dependent on the concentration of the adenovirus DNA binding protein (DBP). However, the molecular basis for the synergistic action of NFI and DBP during replication is at present unknown. We report here that DBP increases the affinity of NFI for its binding site in the replication origin. DBP did not, however, increase the affinity of another eukaryotic sequence-specific DNA binding protein, EBP1, for its recognition site. Other single-stranded DNA binding proteins could not substitute for DBP in increasing NFI affinity for its binding site. In addition, DBP was found to alter the binding kinetics of NFI, both by increasing the rate of association and decreasing the rate of dissociation of NFI with the DNA template. The co-operativity between NFI and DBP was also demonstrated on another DNA template, a human NFI site (FIB2), suggesting that this interaction is of general occurrence and not restricted to the adenovirus origin of replication.

Adenoviridae↗

PubMed1992

Human telomeres are attached to the nuclear matrix.

This report shows that human telomeres are tightly associated with the nuclear matrix. Telomere attachment is observed in several cell types and in all stages of interphase. Mapping experiments show that telomeres are anchored via their TTAGGG repeats; a subtelomeric repeat located immediately proximal to the telomeric TTAGGG repeats is quantitatively released from the nuclear matrix by restriction endonuclease cleavage. TTAGGG repeats introduced at chromosome-internal sites by DNA transfection do not behave as matrix attached loci, suggesting that the telomeric position of the repeats is required for their interaction with the nuclear matrix. These findings are consistent with the idea that telomeres function as a nucleoprotein complex.

Base Sequence↗

PubMed1992

Recognition of the adenovirus type 2 origin of DNA replication by the virally encoded DNA polymerase and preterminal proteins.

Initiation of adenovirus DNA synthesis is preceded by the assembly of a nucleoprotein complex at the origin of DNA replication containing three viral proteins, preterminal protein, DNA polymerase and DNA binding protein, and two cellular proteins, nuclear factors I and III. While sequence specific interactions of the cellular proteins with their cognate sites in the origin of DNA replication are well characterized, the question of how the viral replication proteins recognize the origin has remained unanswered. Preterminal protein and DNA polymerase were therefore purified to homogeneity from recombinant baculovirus infected insect cells. Gel filtration demonstrated that while DNA polymerase existed in monomeric and dimeric forms, preterminal protein was predominantly monomeric and when combined the proteins formed a stable heterodimer. In a gel electrophoresis DNA binding assay each of the protein species recognized DNA within the origin of DNA replication with unique specificity. Competition analysis and DNase I protection experiments revealed that although each protein could recognize the origin, the heterodimer did so with enhanced specificity, protecting bases 8-17 from cleavage with the nuclease. Thus the highly conserved 'core' of the origin of DNA replication, present in all human adenoviruses, is recognized by the preterminal protein--DNA polymerase heterodimer.

Adenoviruses, Human↗

PubMed1994

A new protein domain for binding to DNA through the minor groove.

Protein p6 of the Bacillus subtilis phage phi 29 binds with low sequence specificity to DNA through the minor groove, forming a multimeric nucleoprotein complex that activates the initiation of phi 29 DNA replication. Deletion analysis suggested that the N-terminal part of protein p6, predicted to form an amphipathic alpha-helix, is involved in DNA binding. We have constructed site-directed mutants at the polar side of the putative alpha-helix. DNA binding and activation of initiation of phi 29 DNA replication were impaired in most of the mutant proteins obtained. A 19 amino acid peptide comprising the N-terminus of protein p6 interacted with a DNA fragment containing high-affinity signals for protein p6 binding with approximately 50-fold higher affinity than the peptide corresponding to an inactive mutant. Both wild-type peptide and protein p6 recognized the same sequences in this DNA fragment. This result, together with distamycin competition experiments, suggested that the wild-type peptide also binds to DNA through the minor groove. In addition, CD spectra of the wild-type peptide showed an increase in the alpha-helical content when bound to DNA. All these results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove.

Amino Acid Sequence↗

PubMed1983

Selective in vitro transcription of chloroplast genes.

Transcription of Euglena gracilis chloroplast genes has been investigated by using in vitro transcription systems. A DNA-dependent RNA polymerase responsible for the transcription of rRNA genes has been isolated as a nucleoprotein complex (transcriptionally active chromosome). The RNA polymerase remains tightly bound to the chloroplast DNA template and does not initiate transcription with cloned chloroplast genes. A transcriptionally active extract has been prepared from intact Euglena chloroplasts. The soluble RNA polymerase in this extract recognizes cloned chloroplast tRNA genes and tRNA-sized products have been detected after transcription. The tRNA-sized molecules specifically hybridize to the tRNA genes in the plasmid DNA. At least five tRNA-sized products have been identified from transcription of a trnY1trnH1-trnM1-trnE1-trnW1-trnG1 cluster. Evidence is also presented that processing enzymes in the chloroplast-extract can recognize a polycistronic tRNAVal-tRNAAsn-tRNAArg precursor and process it into tRNA-sized molecules. Truncated templates have been used to demonstrate that the chloroplast tRNA genes are actively transcribed. From a comparison of 5' flanking sequences in chloroplast tRNA genes, a consensus sequence which might function as a promoter, has been identified. The properties of the RNA polymerase involved in the transcription of chloroplast rRNA genes and tRNA genes have been investigated and compared.

Chloroplasts↗

PubMed1995

Varicella-zoster virus assembly protein p32/p36 is present in DNA-containing as well as immature capsids.

Varicella-zoster virus (VZV) produces a group of nucleocapsid proteins (the p32/p36 nucleoprotein complex) which are the VZV analogues of the herpes simplex virus type 1 (HSV-1) and cytomegalovirus (CMV) assembly proteins. There are multiple components in the VZV p32/p36 complex, with major proteins of 32 and 36 kDa and minor proteins of 34 and 38 kDa. In HSV-1 the assembly proteins have been shown to be present in immature (B) capsids, but are removed prior to the formation of mature (C) capsids containing the viral DNA genome. Our work has shown that VZV produces capsids corresponding to the B and C forms. However, in contrast to HSV-1, VZV also produces "B/C" capsids that appear to contain both the assembly proteins and the viral DNA genome. Possible mechanisms for this are discussed. In addition, it was shown that VZV capsids appear to lack the 36 and 38 kDa proteins, and based on this observation we suggest that these may represent unprocessed forms of the assembly protein. In both HSV and CMV, a much larger, cross-reactive protein has been identified as the full-length product of the gene coding for the assembly protein. The homologous VZV gene (ORF 33) theoretically has the capacity to produce a 66 kDa protein. However, no such protein is readily apparent in VZV-infected cells. The presence of an immunoreactive 64 kDa protein was demonstrated in purified VZV capsids which may represent the full-length ORF 33 protein.

Antibodies, Monoclonal↗

PubMed1989

Glutamate receptor agonists increase the expression of Fos, Fra, and AP-1 DNA binding activity in the mammalian brain.

Administration of convulsant doses of Metrazole (pentylenetetrazol) and picrotoxin, as well as maximal electroshock, results in a rapid but transient increase in c-fos mRNA in mouse brain. Elevation of c-fos mRNA is followed by the accumulation and subsequent disappearance of Fos, the protein encoded by c-fos. In addition, immunoblots reveal the induction of two additional proteins that are antigenically related to Fos (Fra, Fos-related antigens). Fos and the various Fra appear and disappear in a staggered manner over an 8 hour period, such that at longer times after stimulation the brain contains no Fos but relatively large amounts of Fra (the latter being designated here by their apparent molecular weights, Fra-46K and Fra-35K). Previous studies have established that Fos, as well as several Fra, contribute to transcription factor AP-1 nucleoprotein complexes along with Jun, the product of the jun proto-oncogene. The appearance in brain of Fos and Fra coincides with a protracted increase in total AP-1 DNA binding activity, indicating that all the Fos-like proteins can participate in AP-1 complexes. Furthermore, the molecular composition of these complexes alters with time after stimulation. The induction of c-fos by Metrazole is blocked or attenuated by known anticonvulsants such as diazepam and valproate as well as the N-methyl-D-aspartate (NMDA) receptor antagonists, 2-amino-5-phosphonovaleric acid (APV) and MK-801. This suggests that fos induction might involve stimulation of a glutamate receptor. This conclusion was strengthened by the observations that two glutamate receptor agonists, kainic acid and NMDA, induced c-fos expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2005

Protamines in the internally fertilizing neobatrachian frog Eleutherodactylus coqui.

The internally fertilizing primitive frog Ascaphus truei (family Ascaphidae) from the Pacific Northwest is the only frog with an intromittent organ. The more advanced neobatrachian frog Eleutherodactylus coqui (family Leptodactylidae) from Puerto Rico has secondarily acquired internal fertilization but mates by cloacal apposition. Nonetheless, both frogs have introsperm with an elongated head containing highly condensed chromatin. Characterization of sperm nuclear basic proteins (SNBPs) in E. coqui by acid-urea polyacrylamide gel electrophoresis indicates that, as in A. truei, testes from a single animal contain several protamines. Amino acid analysis indicates a composition for the most rapidly moving protamine of each species as follows: in E. coqui, ARG (35.6 mol %) + LYS (3.8 mol %) + HIS (7.6 mol %) = 47 mol % total basic residues and in A. truei, ARG (42.1 mol %) + LYS (11.1 mol %) = 53.2 mol % total basic residues. Transmission electron microscopy shows that E. coqui introsperm, like those in A. truei, are elongate with highly condensed chromatin. However, E. coqui introsperm lacks an axial perforatorium that extends into an endonuclear canal. These morphological features are plesiomorphic (primitive) and shared by A. truei with urodeles and basal amniotes (Jamieson et al. (1993) Herpetologica 49:52-65). In E. coqui introsperm, the nucleoprotein complex has a cross-sectional axis of 420 + 20 angstroms and shows a knobby chromatin structural organization in TEM. The presence of arginine-enriched protamines in both a basal anuran like the ascaphid A. truei and a more advanced neobatrachian like the leptodactylid E. coqui supports the hypothesis that internal fertilization acts as a constraint on the range of SNBP diversity in animals.

Amino Acids↗

PubMed2004

Altered expression of the ZBRK1 gene in human breast carcinomas.

The ZBRK1 protein is a member of the KRAB-ZFP family. It functions as a transcriptional repressor by binding to its recognition sequence within target genes and producing a nucleoprotein complex containing its co-repressor BRCA1 and also probably the co-repressor KAP-1. Alterations that affect the ZBRK1 gene have not been reported in human tumours. For this reason, possible alterations in the ZBRK1 gene have been studied by analysing mRNA expression using real-time polymerase chain reaction (PCR), and gene sequence using single strand conformation polymorphism (SSCP) analysis and DNA sequencing, in 61 patients with primary breast carcinomas. BRCA1 mRNA expression and allelic loss were also studied in 25 of the same patients. ZBRK1 was underexpressed in 28 (45.9%) and overexpressed in 18 (29.5%) of the 61 cases. No significant association was observed between ZBRK1 mRNA expression and clinicopathological parameters. Loss of heterozygosity of the BRCA1 gene was found in 3 of 23 (13%) informative cases and BRCA1 mRNA expression was altered in 11 of 25 (44%) cases. No significant correlation was found between altered BRCA1 expression and the different types of ZBRK1 mRNA expression. Nine polymorphisms were found in the ZBRK1 sequence, with no significant differences between patients and control subjects. Altered ZBRK1 expression correlated significantly with two of these polymorphisms. A point mutation in one polymorphism and allelic loss in one patient were also observed, findings that indicate that these inactivation mechanisms do not frequently affect this gene. Altered expression of the ZBRK1 gene is therefore frequent in primary breast carcinoma. The functional significance of the polymorphisms in this gene is unclear.

Adult↗