Chromosomal behavior in starfish (Asterina pectinifera) zygotes under the effect of aphidicolin, an inhibitor of DNA polymerase

When calf thymus histones were labeled fluorescently and microinjected into oocytes of the starfish, Asterina pectinifera, the labeled histones visualized chromosomes during maturation division and cleavage. In doing so, we confirmed the previously reported phenomenon that chromosomes became incompetent at the first cleavage in the aphidicolin-treated egg, although cleavage itself took place. Moreover, we found that chromosomes were aligned at the equator of the metaphase spindle of the first cleavage and that they did not separate into two groups at all, but made a lump in the middle of the spindle. Chromosomes finally entered one blastomere, although they did not participate in the following karyokinesis. DNA and microtubules were examined by cytochemistry and immunofluorescence in order to investigate the relation between chromosome movement and the microtubular cytoskeleton. The mitotic apparatus developed and grew in the aphidicolin-treated cells in the same manner as those in normal cells without normal chromatin condensation or chromosome movement during the first cleavage. However, the mitotic apparatus consisted of two asters without the spindle formed at subsequent cleavages. Electron microscopic study revealed that chromosomes did not condense normally and kinetochores were not detected during the first cleavage. These results indicate that the dynamic changes in microtubular structures during mitosis have poor relation with the chromosome behavior such as prophase chromosome condensation and anaphase chromosome movement.     

Inhibitory effect of Concanavalin A on the cell-to-cell adhesion during early development of the starfish, Asterina pectinifera

The inhibitory effect of Concanavalin A (ConA) on the cell-to-cell adhesion was studied in starfish embryos. ConA reversibly blocked the formation of intercellular adhesion in embryos denuded of fertilization membrane as well as in normal embryos, without affecting cell division and thereby inhibiting the morphogenetic movement of blastulation. A large dose of ConA dissociated both denuded and normal embryos to single cells at blastula and gastrula stage. Succinyl ConA (Suc-ConA) has the same effect on cell-to-cell adhesion, though critical concentration was slightly higher than that of ConA. These effects of ConA or Suc-ConA were prevented by α-methyl- -mannoside (αMM). Study of the binding of fluorescein-conjugated ConA to the cell surface showed that ConA receptors were present in the surface of fertilized egg and cells at all stages examined. These findings suggest that ConA receptors play an important role in cell-to-cell adhesion during the early morphogenesis of starfish.     

Selective abrogation of alloreactivity via priming in the presence of aphidicolin, a specific inhibitor of DNA polymerase

Aphidicolin, a specific and direct inhibitor of eukaryotic DNA polymerase alpha, was used to investigate its impact on immunologic reactions in vitro. Dose response curve of the inhibitory effect was studied in murine and human primary allogeneic responses, as well as the proliferative responses to both PHA and Con A mitogens. The presence of aphidicolin during the allosensitization phase in secondary MLR of mice splenocytes resulted in complete abolishment of the subsequent response directed against the priming alloantigens, whereas alloreactivity to unrelated alloantigen-bearing cells was inhibited to a much lesser degree. The allosensitized aphidicolin-treated cells lost the ability to respond to subsequent PHA stimulation, but were capable of exerting a high responsiveness to Con A. The presence of aphidicolin during the allosensitization phase in secondary MLR of human mononuclear cells resulted in markedly decreased alloreactivity directed against the priming cells, but spared the subsequent response to unrelated alloantigens and to both PHA and Con A mitogenic stimuli. It is suggested that aphidicolin may be used for selective inactivation of proliferating cells without interfering with immunologic functions of other quiescent subsets. Aphidicolin may thus be a useful agent for induction of specific unresponsiveness in experimental models of allogeneic transplantation.     

The effects of aphidicolin,an inhibitor of DNA replication,on sea urchin development

Aphidicolin, an inhibitor of DNA polymerase α, arrests DNA synthesis without affecting RNA and protein synthesis at all stages of sea urchin development. Cleavage is quickly stopped and hatching is prevented by the presence of the drug at 2 μg/ml. Treatment with aphidicolin of young blastulae prevents gastrulation, but inhibition of archenteron invagination and skeleton formation is incomplete when the drug is added to late blastulae or early gastrulae. The role of cell division in gastrulation is discussed.     

3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, is a stimulator, not an inhibitor, of DNA repair

An inhibitor of poly(ADP-ribose) synthesis, 3-aminobenzamide (3AB), at low concentrations (0.01-0.1 mM) was found to reduce strand-break frequencies and increase repair replication in human lymphoid cells damaged by methyl methanesulfonate. A concentration of 0.1 mM 3AB was adequate to produce a maximum effect on strand-break frequencies and repair replication. This evidence, together with our previous measurements, demonstrates that 3AB cannot be regarded as an inhibitor of DNA repair; rather, it actually accelerates the ligation of DNA repair patches. Previous considerations of 3AB as a repair inhibitor may have derived from the use of excessive concentrations above 1 mM that may have stimulated additional damage and from the use of ethyl alcohol as a solvent for 3AB. Interpretations of the role of single-strand breaks and poly(ADP-ribose) in DNA repair, differentiation, and gene activity may need reevaluation because they have frequently been based on an erroneous notion of 3AB as a repair inhibitor, when its mode of action is, in fact, more complex.     

Sensitivity of halobacteria to aphidicolin, an inhibitor of eukaryotic α-type DNA polymerases

Abstract An Escherichia coli dnaAts mutation is integratively suppressed by pBR322-derived plasmids carrying DNA inserts homologous to the bacterial chromosome. The findings that the relation between the length of the homologous region and the level of integrative suppression is linear, allows to calculate the extent of homology between the bacterial chromosome and any DNA fragment inserted into pBR322.     

DNA Sequence, Chromosomal Localization, and Tissue Expression of the Mouse Proteasome SubunitLmp10(Psmb10) Gene

Proteasomes are nonlysosomal multicatalytic proteases involved in antigen processing. Three of the 10 mammalian proteasome β subunits (LMP2, LMP7, and LMP10) are induced by IFN-γ. Two of these (LMP2 and LMP7) are encoded in the major histocompatibility complex of both human (chromosome 6) and mouse (chromosome 17). However, the human homologue ofLmp10, MECL1,is found on chromosome 16. Here we show that in mice,Lmp10is a single-copy gene localized to chromosome 8, in a region of conserved synteny with human chromosome 16. Sequencing of a 129/SvJ strain genomic clone revealed that the gene has eight exons spanning 2.3 kb. Characterization of a full-length mouse cDNA clone indicates thatLmp10encodes a protein of 273 amino acids with a calculated molecular weight of 29 kDa and an isoelectric point of 6.86. Northern analysis ofLmp2, Lmp7,andLmp10showed expression in heart, liver, thymus, lung, and spleen, but not in brain, kidney, skeletal muscle, or testis.     

Mode of inhibition of the DNA polymerase of Methanococcus vannielii by aphidicolin

The mode of action of aphidicolin on DNA synthesis catalysed by the DNA polymerase of Methanococcus vannielii is competitive for dCTP, noncompetitive for dATP, dGTP and dTTP and uncompetitive for activated DNA. The kinetic data are accounted for by a mechanism in which dCTP and aphidicolin compete for the dCTP-specific binding site on the DNA polymerase. The dissociation constant for the aphidicolin--DNA-polymerase complex is 0.04-0.07 microM. Similar modes of inhibition of DNA synthesis exist for DNA polymerase alpha of higher eucaryotes but not for eubacteria or viruses and suggests a close functional relationship between the DNA polymerase of eucaryotes and of the archaebacterium M. vannielii.     

Mutations in the herpes simplex virus DNA polymerase gene conferring hypersensitivity to aphidicolin.

Fourteen mutants known or likely to contain mutations in the herpes simplex virus DNA polymerase gene were examined for their sensitivity to aphidicolin in plaque reduction assays. Eleven of these exhibited some degree of hypersensitivity to the drug; altered aphidicolin-sensitivity correlated with altered sensitivity to the pyrophosphate analog, phosphonoacetic acid. The DNA polymerase specified by one of these mutants, PAAr5, required roughly seven-fold less aphidicolin to inhibit its activity by 50% than did polymerase specified by its parental strain. Mutations responsible for the aphidicolin-hypersensitivity phenotype of PAAr5 were mapped to an 0.8 kbp region in the herpes simplex virus DNA polymerase locus. These data taken together indicate that 1) mutations in the herpes simplex virus DNA polymerase gene can confer altered sensitivity to aphidicolin, 2) that the HSV polymerase is sensitive to aphidicolin in vivo, and 3) that amino acid alterations which affect aphidicolin binding may affect the pyrophosphate exchange-release site as well, suggesting that aphidicolin binds in close proximity to this site.     

Plasmodium falciparum: synchronization of asexual development with aphidicolin, a DNA synthesis inhibitor

The asexual development cycle of Plasmodium falciparum, a malarial parasite of humans, has been synchronized in culture by treating ring-stage parasites with aphidicolin, an inhibitor of DNA synthesis. Optimization of both the concentration of drug added to ring stage containing red blood cells and the duration of exposure of parasites to drug led to a reversible block of their maturation at the early trophozoite stage. Release of the aphidicolin block led to a synchronous development of parasites that was manifested by about 80% of the new ring stages being produced within a 2- to 3-hr interval.     

Fertilization of the starfish Astropecten aurantiacus

In the starfish Astropecten aurantiacus the acrosome reaction occurs when the spermatozoon contacts the outer surface of the jelly layer. A long thin acrosomal filament is extruded from the anterior region of the spermatozoon and establishes contact with the oocyte surface. This latter interaction initiates the movement of the spermatozoon to the oocyte surface, formation of the fertilization cone and the cortical reaction. The first detectable electrical change across the oocyte plasma membrane during interaction with the spermatozoon is the fertilization potential (FP) which occurs simultaneously with the cortical reaction. The FP is probably the electrical result of the modification of the oocyte plasma membrane during cortical exocytosis. There are no primary step-like depolarizations during fertilization of starfish oocytes, which contrasts with the situation in sea urchin eggs [see 13]. We suggest that the difference in electrical response to fertilization of starfish oocytes and sea urchin eggs may be attributed to the location of the acrosome reaction in these animals and not to their different meiotic states.     

Isolation of a DNA polymerase I (polA) mutant of Rhizobium leguminosarum that has significantly reduced levels of an IncQ-group plasmid

A population of Tn5 mutagenised Rhizobium leguminosarum cells was screened for mutants affected in protein secretion by introducing a plasmid carrying the Erwinia chrysanthemi prtB gene and screening for mutants defective in secretion of the protease PrtB. One such mutant (A301) also appeared to be defective in secretion of the R. leguminosarum nodulation protein NodO. Genetic analysis showed that the defect in A301 was caused by the Tn5 insertion. However the DNA sequence adjacent to the site of Tn5 insertion had significant homology to the Escherichia coli polA gene, which encodes DNA polymerase I. The mutant A301 showed increased sensitivity to ultraviolet light, a characteristic of polA mutants of E. coli. The apparent defect in secretion by A301 was due to a large decrease in the copy number of the IncQ group replicon on which prtB and nodO were cloned and this decreased the total amounts of PrtB or NodO protein synthesised and secreted by the polA mutant. The polA mutant had a lower growth rate than the parent strain on both rich and minimal media, but there was no obvious effect of the polA mutation on the symbiosis of R. leguminosarum bv. viciae with pea.     

Isolation of protoplasts from zygotes of Fucus distichus (L.) powell (Phaeophyta)

Two-step procedures were developed for the isolation of protoplasts from 6-hour-old zygotes of Fucus distichus L., using commercial cellulases and alginate-lyases from marine molluscs. Protoplasts were obtained either as a suspension or attached to a substratum. Protoplasts yields were greater than 95% of the cell population and over 80% regenerated a wall, germinated and divided into a polar, multicellular embryo. Zygote development from regenerated protoplasts was also quite synchronous. The same procedures were less effective in removing the cell wall from zygotes older than 8 h, and did not yield significant numbers of protoplasts from two-celled embryos.     

Site of the base change in the vaccinia virus DNA polymerase gene which confers aphidicolin resistance.

An aphidicolin-resistant mutant of vaccinia virus has been shown to encode an altered viral DNA polymerase that is more resistant to aphidicolin. Marker transfer experiments with the DNA from the resistant virus localized the mutation site to an RsaI segment within the portion of the HindIII-E segment which has been shown to contain the viral DNA polymerase gene. Nucleotide sequence analysis of the mutant DNA showed a single GC to AT transition at position 2430, which indicates a leucine-to-methionine change at residue 645 in the protein.     

A DNA polymerase alpha partially resistant to aphidicolin in cells and embryos of Med-fly (Ceratitis capitata Wied.)

We have studied the DNA polymerase activities in cultured cells and embryos of Med-fly (Ceratitis capitata Wied.) and we observed that only DNA polymerases alpha and gamma were detectable in crude extracts. The level of DNA polymerase alpha, measured during the life cycle of the insect embryos, increased in parallel with the rate of embryonic cell proliferation, whereas DNA polymerase gamma increased at much later fertilization time, when cell differentiation is already taking place. DNA polymerase alpha, purified 100 folds from Med-fly embryos, was 10 times more resistant to aphidicolin, its specific inhibitor, than the mammalian DNA polymerase alpha. In situ visualization of the active peptides after NaDodSO4/PAGE, confirmed that only high Mr DNA polymerase fragments were present in embryo extracts and in purified preparations of DNA polymerase alpha. It appears that C. capitata cells represent a rather peculiar system in the phylogeny of DNA polymerases since they are devoid of DNA polymerase beta and present a DNA polymerase alpha partially resistant to aphidicolin.     

Dynamics of DNA polymerase I (Klenow fragment) under external force

During DNA synthesis, high-fidelity DNA polymerase (DNAP) translocates processively along the template by utilizing the chemical energy from nucleotide incorporation. Thus, understanding the chemomechanical coupling mechanism and the effect of external mechanical force on replication velocity are the most fundamental issues for high-fidelity DNAP. Here, based on our proposed model, we take Klenow fragment as an example to study theoretically the dynamics of high-fidelity DNAPs such as the replication velocity versus different types of external force, i.e., a stretching force on the template, a backward force on the enzyme and a forward force on the enzyme. Replication velocity as a function of the template tension with only one adjustable parameter is in good agreement with the available experimental data. The replication velocity is nearly independent of the forward force, even at very low dNTP concentration. By contrast, the backward force has a large effect on the replication velocity, especially at high dNTP concentration. A small backward force can increase the replication velocity and an optimal backward force exists at which the replication velocity has maximum value; with any further increase in the backward force the velocity decreases rapidly. These results can be tested easily by future experiments and are aid our understanding of the chemomechanical coupling mechanism and polymerization dynamics of high-fidelity DNAP.