Characterization of a potent catenation activity of HeLa cell nuclei

Using an assay which measures catenation of a supercoiled DNA template, we have characterized and quantitated a potent activity identified in crude fractions of HeLa cell nuclei. Catenation requires Mg-ATP and a DNA-condensing agent, polyvinyl alcohol. A filter-binding or agarose gel assay can be used to quantitate activity. In this reaction, DNA topoisomerase I relaxes the input supercoiled DNA to provide DNA topoisomerase II, a strongly favored template for catenation. DNA topoisomerase II preferentially catenates relaxed DNA over supercoiled DNA by a factor of 100. One molecule of DNA topoisomerase II is able to catenate about 20 circles of relaxed DNA/min at 30 degrees C but only 0.16 circle of supercoiled DNA/min at 30 degrees C. The purified HeLa topoisomerase I can also catenate DNA under these assay conditions, yet in an ATP-independent fashion. It is much less efficient than topoisomerase II; one molecule of topoisomerase I catenates only about 3.8 X 10(-3) molecules of supercoiled DNA/min at 30 degrees C with a DNA template containing 5% nicked circles. This remarkable difference between the two enzymes allows quantitation of DNA topoisomerase II activity seen in the presence of excess topoisomerase I. Unlike Escherichia coli topoisomerase I (omega), catenation by the HeLa topoisomerase I is not stimulated by gapped circles.     

A simple method for cloning and replica plating of mammalian cells using multicellular spheroids.

V 79/4 Chinese hamster cells or HeLa cells grow in Eagle's MEM supplemented with 25 microgram/ml dextran sulphate to form clonal multicellular spheroids. These cell clones, consisting of 5-10(2) cells, are easy to separate, to transfer from one culture vessel into another and grow as normal monolayer colonies on Dederon cloth circles after subculture in Eagle's MEM without dextran sulphate. A simple replica technique is described by which 500 clones can be transfered onto at least 3 replica cloth circles, 10 cm in diameter, with a replica plating efficiency of approximately 100%.     

Cyclophilin A-dependent restriction of human immunodeficiency virus type 1 capsid mutants for infection of nondividing cells

Among retroviruses, lentiviruses are unusual in their ability to efficiently infect both dividing and nondividing cells, such as activated T cells and macrophages, respectively. Recent studies implicate the viral capsid protein (CA) as a key determinant of cell-cycle-independent infection by human immunodeficiency virus type 1 (HIV-1). We investigated the effects of the host cell protein cyclophilin A (CypA), which binds to HIV-1 CA, on HIV-1 infection of nondividing cells. The HIV-1 CA mutants A92E, T54A, and R132K were impaired for infection of aphidicolin-arrested HeLa cells, but not HOS cells. The mutants synthesized normal quantities of two-long-terminal-repeat circles in arrested HeLa cells, indicating that the mutant preintegration complexes can enter the nuclei of both dividing and nondividing cells. The impaired infectivity of the CA mutants on both dividing and nondividing HeLa cells was relieved by either pharmacological or genetic disruption of the CypA-CA interaction or by RNA interference-mediated depletion of CypA expression in target cells. A second-site suppressor of the CypA-restricted phenotype also restored the ability of CypA-restricted HIV-1 mutants to infect growth-arrested HeLa cells. These results indicate that CypA-restricted mutants are specifically impaired at a step between nuclear import and integration in nondividing HeLa cells. This study reveals a novel target cell-specific restriction of HIV-1 CA mutants in nondividing cells that is dependent on CypA-CA interactions.     

A homogeneous type II DNA topoisomerase from HeLa cell nuclei

Using kinetoplast DNA networks as a substrate in a decatenation assay, we have purified to apparent homogeneity a type II DNA topoisomerase from HeLa cell nuclei. The most pure preparations contain a single polypeptide of 172,000 daltons as determined by sodium dodecyl sulfate-gel electrophoresis. The molecular weight of the native protein, based on sedimentation and gel filtration analyses, is estimated to be 309,000. These results suggest that the enzyme is a dimer of 172,0090-dalton subunits. The enzyme is a type II topoisomerase as demonstrated by its ability to change the linking number of DNA circles in steps of two and to decatenate or unknot covalently closed DNA circles. No gyrase activity is detectable. ATP is required for the relaxation, decatenation, and unknotting of DNA, and a DNA-dependent ATPase activity is present in the most pure fractions. ATP is hydrolyzed to ADP in this properties to T4 DNA topoisomerase (Liu, L. F., Liu, C. C., and Alberts, B. M. (1979) Nature 281, 456-461).     

RNA 3'-terminal phosphate cyclase activity and RNA ligation in HeLa cell extract.

HeLa cell extract contains RNA ligase activity that converts linear polyribonucleotides to covalently closed circles. RNA substrates containing 2',3'-cyclic phosphate and 5'-hydroxyl termini are circularized by formation of a normal 3',5' phosphodiester bond. This activity differs from a previously described wheat germ RNA ligase which circularizes molecules with 2',3'-cyclic and 5' phosphate ends by a 2'-phosphomonester, 3',5'-phosphodiester linkage (Konarska et al., Nature 293, 112-116, 1981; Proc. Natl. Acad. Sci. USA 79, 1474-1478, 1982). The HeLa cell ligase can also utilize molecules with 3'-phosphate ends. However, in this case ligation is preceded by an ATP-dependent conversion of the 3'-terminal phosphate to the 2',3' cyclic form by a novel activity, RNA 3'-terminal phosphate cyclase. Both RNA ligase and RNA 3'-terminal phosphate cyclase activities are also present in extract of Xenopus oocyte nuclei, consistent with a role in RNA processing.     

Human epithelial cell intermediate filaments: isolation, purification, and characterization

Intermediate filaments (IF) isolated from human epithelial cells (HeLa) can be disassembled in 8 M urea and reassembled in phosphate-buffered solutions containing greater than 0.1 mg/ml IF protein. Eight proteins were associated with HeLa IF after several disassembly-reassembly cycles as determined by sodium dodecyl sulfate gel electrophoresis (SDS PAGE). A rabbit antiserum directed against HeLa IF contained antibodies to most of these proteins. The immunofluorescence pattern that was seen in HeLa cells with this antiserum is complex. It consisted of a juxtanuclear accumulation of IF protein and a weblike array of cytoplasmic fibers extending to the cell border. Following preadsorption with individual HeLa IF proteins, the immunofluorescence pattern in HeLa cells was altered to suggest the presence of at least two distinct IF networks. The amino acid composition and alpha-helix content (approximately 38%) of HeLa IF proteins was similar to the values obtained for other IF proteins. One-dimensional peptide maps show extensive homology between the major HeLa IF protein of 55,000-mol- wt and a similar 55,000-mol-wt protein obtained from hamster fibroblasts (BHK-21). HeLa 55,000-mol-wt homopolymer IF assembled under conditions similar to those required for BHK-21 55,000-mol-wt homopolymers. Several other proteins present in HeLa IF preparations may be keratin-like structural proteins. The results obtained in these studies indicate that the major HeLa IF protein is the same major IF structural protein found in fibroblasts. Ultrastructural studies of HeLa cells revealed two distinct IF organizational stages including bundles and loose arrays. In addition, in vitro reconstituted HeLa IF also exhibited these two organizational states.     

Experiments Testing the Causes of Namibian Fairy Circles

The grasslands on the sandy soils of the eastern edge of the Namib Desert of Namibia are strikingly punctuated by millions of mostly regularly-spaced circular bare spots 2 to 10 m or more in diameter, generally with a margin of taller grasses. The causes of these so called fairy circles are unknown, but several hypotheses have been advanced. In October 2009, we set up experiments that specifically tested four hypothesized causes, and monitored these 5 times between 2009 and 2015. Grass exclusion in circles due to seepage of subterranean vapors or gases was tested by burying an impermeable barrier beneath fairy circles, but seedling density and growth did not differ from barrier-less controls. Plant germination and growth inhibition by allelochemicals or nutrient deficiencies in fairy circle soils were tested by transferring fairy circle soil to artificially cleared circles in the grassy matrix, and matrix soil to fairy circles (along with circle to circle and matrix to matrix controls). None of the transfers changed the seedling density and growth from the control reference conditions. Limitation of plant growth due to micronutrient depletion within fairy circles was tested by supplementing circles with a micronutrient mixture, but did not result in differences in plant seedling density and growth. Short-range vegetation competitive feedbacks were tested by creating artificially-cleared circles of 2 or 4 m diameter located 2 or 6 m from a natural fairy circle. The natural circles remained bare and the artificial circles revegetated. These four experiments provided evidence that fairy circles were not caused by subterranean vapors, that fairy circle soil per se did not inhibit plant growth, and that the circles were not caused by micronutrient deficiency. There was also no evidence that vegetative feedbacks affected fairy circles on a 2 to 10 m scale. Landscape-scale vegetative self-organization is discussed as a more likely cause of fairy circles.     

DNA circles with cruciforms from Isospora (Toxoplasma) gondii

We have isolated a closed circular duplex DNA fraction from the unicellular parasite Isospora (Toxoplasma) gondii and examined the purified DNA by electron microscopy. A major part of this circular DNA consists of 12-micron circles containing a cruciform with 0.5-micron tails. We also found 23-micron circles with the properties expected of head-to-tail dimers of the 12-micron circles. Some of these dimers have two cruciforms with 0.4-micron tails, some have one cruciform with 0.8-micron tails. When ethidium bromide was diffused into the DNA solution, circles with tails were replaced by twisted circles without tails. Direct mixing of the DNA with high ethidium bromide concentrations (5 micrograms/ml) gave rise to highly twisted circles with tails. This proves that the tailed circles are covalently continuous and indicates that ethidium bromide blocks branch migration. The 0.5-micron tails are part of a 1.7-micron palindrome, which was visualized by spreading denatured DNA under snap-back conditions. We argue that the cruciform is not present in vivo and that the 12-micron circles may represent the mitochondrial DNA of Toxoplasma.     

Microtubule-associated proteins of HeLa cells: heat stability of the 200,000 mol wt HeLa MAPs and detection of the presence of MAP-2 in HeLa cell extracts and cycled microtubules

One of the major groups of microtubule-associated proteins (MAPs) found associated with the microtubules isolated from HeLa cells has a molecular weight of just over 200,000. Previous work has demonstrated that these heLa MAPs are similar in several properties to MAP-2, one of the major MAPs of mammalian neural microtubules, although the two types of proteins are immunologically distinct. The 200,000 mol wt HeLa MAPs have now been found to remain soluble after incubation in a boiling water bath and to retain the ability to promote tubulin polymerization after this treatment, two unusual properties also shown by neural MAP- 2. This property of heat stability has allowed the development of a simplified procedure for purification of the 200,000 HeLa MAPs and has provided a means for detection of these proteins, even in crude cell extracts. These studies have also led to the detection of a protein in crude extracts of HeLa cells and in cycled HeLa microtubules which has been identified as MAP-2 on the basis of (a) comigration with calf brain MAP-2 on SDS PAGE, (b) presence in purified microtubules, (c) heat stability, and (d) reaction with two types of antibodies prepared against neural high molecular weight-MAPs, one of these a monoclonal antibody against hog brain MAP-2, although present in HeLa cells, is at all stages of microtubule purification a relatively minor component in comparison to the 200,000 HeLa MAP's.     

A correlation between the effects of calcium on intercellular adhesion and electrostatic repulsion between cells.

Calcium ions enchance the mutual adhesiveness of HeLa cells harvested from suspension cultures in which growth is density inhibited. No significant effect of calcium is observed on the mutual adhesiveness of HeLa cells from fast growing suspension cultures. Agglutinative titration of the cells using poly-L-lysine, mol. wt 15000, shows that calcium ions reduce the strength of the repulsive forces on density inhibited HeLa cells. The agglutination curve of the nonrepulsive fast growing HeLa cells is not significantly modified by the addition of calcium. The results support the conclusion that the effect of calcium on the mutual adhesiveness of density inhibited cells is due to a weakening of the repulsive forces on these cells.     

Replicating circular DNA molecules in yeast

The yeast Saccharomyces cerevisiae contains a class of small circular DNA molecules, approximately 2 μm in contour length (Sinclair et al., 1967). In this report, it is shown that these molecules replicate as double-branched circles, similar to those observed during replication of the bacteriophage λ and Escherichia coli chromosomes. A normal rate of replication of these DNA circles requires the function of a nuclear gene, cdc 8.     

The H circles of Leishmania tarentolae are a unique amplifiable system of oligomeric DNAs associated with drug resistance

We have induced drug resistance against methotrexate, an inhibitor of dihydrofolate reductase, and CB3717, an inhibitor of thymidylate synthetase in a strain of Leishmania tarentolae. The drug-resistant strains contain extrachromosomal DNA circles of 68 kilobases with a 30-kilobase inverted duplication flanked by 4- and 5 kilobase unique segments. We show that these circles are highly homologous to the drug-induced H circles of L. tropica (1). All three L. tarentolae strains analyzed contain a chromosomal copy of the H region without duplication, but two of the three strains contain extrachromosomal H circles as well, predominantly present as H circle dimers in one strain and as tetramers in the other. After induction of methotrexate resistance, monomeric circles, presumably derived from the oligomers, become the major type of circle. Our results indicate that the H region represents a genomic region that can be copied at very low frequency to yield circles by a precise, but unusual mechanism under natural conditions in wild-type cells. Although superficially analogous to the episomes of bacteria, the system is without precedent in nature.     

In vitro replication directed by a cloned adenovirus origin

A 5.7-kb recombinant plasmid, called XD-7, contains the terminal XbaI-E fragment from the left end of type 2 adenovirus cloned into the EcoRI site of pBR322. An average of 9% +/- 1% of input supercoiled, protein-free XD-7 DNA replicated as rolling circles with single-stranded tails ranging up to unit length and longer in reaction mixtures containing nuclear and cytoplasmic extracts from adenovirus-infected, but not uninfected, HeLa cells. The adenovirus origin was mapped on XD-7 by electron microscopy at the left boundary of the cloned adenovirus segment. Since replication proceeded rightwards, we conclude that the adenovirus l strand was displaced during replication. No origin was located at or near the EcoRI site on pBR322. Reversing the orientation of the adenovirus origin reversed the direction of replication, and deletion of the adenovirus origin abolished replication.     

Edaphic properties enable facilitative and competitive interactions resulting in fairy circle formation

Millions of generally regularly spaced, roughly circular barren patches called fairy circles occur in a narrow band ca 100 km inland of the south‐west African coast. These generally have conspicuously taller peripheral grasses in a shorter grass matrix. The origins of these fairy circles are controversial, but one possibility is that they are self‐organizing emergent vegetation patterns that are the consequence of interplay between positive (facilitative) and negative (competitive) interactions between grasses. We hypothesized that the coarse textured sand on which fairy circles occur creates a hydraulically and nutritionally connected landscape, in which neighbouring fairy circles competitively influence each other over several metres, while providing opportunity for focusing of resources around the peripheral grasses. To test our hypotheses we conducted three main groups of analyses: 1) we measured grass biomass to assess facilitative and competitive effects of the component grasses; 2) across a region with fairy circles we measured the size and density of fairy circles and correlated that with water infiltration rates into soil; 3) we measured the capacity of soil to conduct water pulses and ¹⁵N tracers. We found evidence of facilitative interactions in the periphery of the fairy circles and competitive suppression of the matrix grass proximal to the periphery. Across the region, fairy circle size was positively correlated with soil infiltration rates and negatively with precipitation. This suggests that fairy circles emerge in soils with high capacity for water flux that enables landscape hydraulic connectivity. Water‐ and ¹⁵N‐pulse experiments showed that edaphic resources were highly mobile, moving up to 7.5 m over a period of 1–3 weeks. We concluded that the evidence is consistent with an emergent vegetation pattern explanation for the origins of fairy circles and that the circles are more closely associated with a highly connective edaphic environment, rather than with particular biota.     

Isolation and partial characterization of a cage of filaments that surrounds the mammalian mitotic spindle

Mitotic cells have been detergent extracted under conditions that support microtubule assembly. When HeLa cells are lysed in the presence of brain tubulin, mitotic-arrested cells nucleate large asters and true metaphase cells yield spindles that remain enclosed within a roughly spherical cage of filamentous material. Detergent-extracted mitotic Chinese hamster ovary (CHO) cells show a similar, insoluble cage but the mitotic apparatus is only occasionally stabilized. In later stages of mitosis, HeLa cages are observed in elongated and furrowed configurations. In the terminal stages of cell division, two daughter filamentous networks are connected by the intercellular bridge. When observed in the electron microscope the cages include fibers 7-11 nm in diameter. The polypeptide composition of cages isolated from mitotic HeLa cells is complex, but the major polypeptides are a group with mol wt ranging from 43,000-60,000 daltons and a high molecular weight polypeptide. CHO cells contain a subset of these proteins which includes a major 58,000-dalton and a high molecular weight polypeptide. Two different antisera directed against the vimentin-containing intermediate filaments bind to polypeptides in the electrophoretic profiles of isolated HeLa and CHO cages and stain the cages, as visualized by indirect immunofluorescence. These results suggest that the HeLa and CHO cages include intermediate filaments of the vimentin type. The polypeptide composition of HeLa cages suggests that they also contain tonofilaments. The cages apparently form as the cells enter mitosis. We propose that these filamentous cages maintain the structural continuity of the cytoplasm while the cell is in mitosis.     

Zinc adaptation and resistance to cadmium toxicity in mammalian cells: molecular insight by proteomic analysis

To identify proteins involved in cellular adaptive responses to zinc, a comparative proteome analysis between a previously developed high zinc- and cadmium-resistant human epithelial cell line (high zinc-resistant HeLa cells, HZR) and the parental HeLa cells has been carried out. Differentially produced proteins included cochaperones, proteins associated with oxido-reductase activities, and ubiquitin. Biochemical pathways to which these proteins belong were probed for their involvement in the resistance of both cell lines against cadmium toxicity. Among ER stressors, thapsigargin sensitized HZR cells, but not HeLa cells, to cadmium toxicity more acutely than tunicamycin, implying that these cells heavily relied on proper intracellular calcium distribution. The similar sensitivity of both HeLa and HZR cells to inhibitors of the proteasome, such as MG-132 or lactacystin, excluded improved proteasome activity as a mechanism associated with zinc adaptation of HZR cells. The enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) was overproduced in HZR cells as compared to HeLa cells. It transforms HPP to homogentisate in the second step of tyrosine catabolism. Inhibition of HPPD decreased the resistance of HZR cells against cadmium, but not that of HeLa cells, suggesting that adaptation to zinc overload and increased HPP removal are linked in HZR cells.     

Circularization of human immunodeficiency virus type 1 DNA in vitro.

Linear viral DNA present in cytoplasmic extracts of cells newly infected with human immunodeficiency virus type 1 can be induced to form 1-LTR and 2-LTR circles by incubation of the extracts in the presence of added nucleoside triphosphates. No circular DNA forms are detected when extracts are incubated in the absence of added nucleoside triphosphates. Restriction enzyme analysis and polymerase chain reaction analysis with selected primers, as well as DNA sequence analysis of the polymerase chain reaction products, show that most of the 2-LTR circles are the result of autointegration reactions, while 1-LTR circles result from recombination between the long terminal repeats on the linear viral DNA. In addition, a small amount of simple 2-LTR circles, formed by end-to-end joining of the linear viral DNA, is formed in vitro. Integration of the linear viral DNA into heterologous DNA competes effectively with the formation of 2-LTR circles by autointegration. However, concentrations of target DNA which completely block autointegration have no effect on the formation of 1-LTR circles or simple 2-LTR circles. Factors present in extracts of uninfected cells can mediate the formation of 1-LTR circles and simple 2-LTR circles from purified deproteinated linear viral DNA, indicating that viral proteins are not necessary for the formation of these two types of circular viral DNA. These experiments demonstrate that all the transformations of linear viral DNA which occur in the nuclei of cells infected with human immunodeficiency virus type 1 can be reproduced in vitro.