Phylogeny of the Large Extrachromosomal DNA of Organisms in the Phylum Apicomplexa

ABSTRACT. Organisms in the phylum Apicomplexa appear to have a large extrachromosomal DNA which is unrelated to the mitochondrial DNA. Based on the apparent gene content of the large (35 kb) extrachromosomal DNA of Plasmodium falciparum , it has been suggested that it is a plastid-like DNA, which may be related to the plastid DNA of rhodophytes. However, phylogenetic analyses have been inconclusive. It has been suggested that this is due to the unusually high A + T content of the Plasmodium falciparum large extrachromosomal DNA. To further investigate the evolution of the apicomplexan large extrachromosomal DNA, the DNA sequence of the organellar ribosomal RNA gene from Toxoplasma gondii , was determined. The Toxoplasma gondii rDNA sequence was most similar to the large extrachromosomal rDNA of Plasmodium falciparum , but was much less A + T rich. Phylogenetic analyses were carried out using the LogDet transformation to minimize the impact of nucleotide bias. These studies support the evolutionary relatedness of the Toxoplasma gondii rDNA with the large extrachromosomal rDNA of Plasmodium falciparum and with the organellar rDNA of another parasite in the phylum Apicomplexa, Babesia bovis. These analyses also suggest that the apicomplexan large extra-chromosomal DNA may be more closely related to the plastid DNA of euglenoids than to those of rhodophytes.     

Nucleotide Sequence Analysis of DNA

There are three major obstacles to the analysis of the nucleotide sequence in a DNA molecule starting from a known location in the DNA molecule. First, it is difficult to obtain large quantities of homogeneous DNA. Second, even the smallest DNA molecules contain several thousand nucleotides which make sequence analysis prohibitive. Third, there are no highly base-specific DNAases available for degrading DNA for sequence analysis. We have overcome some of these obstacles; first, by incorporating highly labelled deoxynucleotides into DNA in vitro, small amounts of material can be used for sequence analysis. Second, the nucleotide sequence of DNA molecules can now be determined from the 5′-terminal. Thus, two dodecanucleotide sequences corresponding to the two cohesive ends of λ DNA have been determined¹ and a nona-decanucleotide sequence corresponding to one cohesive end of phage 186 DNA has been completed². So far, our approach is limited to starting the analysis from the 5′-ends of a DNA molecule. A more general approach is being developed for starting the analysis from other selected parts of a DNA molecule with the use of specifically designed primers.     

Characterization of yeast artificial chromosomes from Plasmodium falciparum: construction of a stable, representative library and cloning of telomeric DNA fragments.

Molecular genetic studies of the human malaria parasite Plasmodium falciparum have been hampered in part due to difficulties in stably cloning and propagating parasite genomic DNA in bacteria. This is thought to be a result of the unusual A+T bias (>80%) in the parasite's DNA. Pulsed-field gel electrophoretic separation of P. falciparum chromosomes has shown that large chromosomal polymorphisms, resulting from the deletion of DNA from chromosome ends, frequently occur. Understanding the biological implications of this chromosomal polymorphism will require the analysis of large regions of genomic, and in particular telomeric, DNA. To overcome the limitations of cloning parasite DNA in bacteria, we have cloned genomic DNA from the P. falciparum strain FCR3 in yeast as artificial chromosomes. A pYAC4 library with an average insert size of approximately 100 kb was established and found to have a three to fourfold redundancy for single-copy genes. Unlike bacterial hosts, yeast stably maintain and propagate large tracts of parasite DNA. Long-range restriction enzyme mapping of YAC clones demonstrates that the cloned DNA is contiguous and identical to the native parasite genomic DNA. Since the telomeric ends of chromosomes are underrepresented in YAC libraries, we have enriched for these sequences by cloning P. falciparum telomeric DNA fragments (from 40 to 130 kb) as YACs by complementation in yeast.     

DNA polymerase from Sulfolobus acidocaldarius. Replication at high temperature of long stretches of single-stranded DNA

The activity of a homogeneous DNA polymerase from the thermophilic archaebacterium, Sulfolobus acidocaldarius, on a singly primed, single-stranded recombinant phage M13 DNA has been examined. At the optimal temperature (70 to 75 degrees C) this template is efficiently replicated in ten minutes using a ratio of enzyme molecule to primed-template of 0.8. Analysis of DNA products during the course of polymerization shows that species of quite homogeneous size are observed and that the number of primers extended by the enzyme is constant, whatever the enzyme molecule to primed template ratio is in the range 1/50 to 2, indicating that the 100 x 10(3) Mr DNA polymerase from S. acidocaldarius is randomly recycled on the template molecules. At non-optimal temperature (60 degrees C and 80 degrees C) the distribution of products observed indicated the presence of arrest sequences; some have been shown to be reversible. One of these pausing signals detected at 80 degrees C has been further analysed, and has been found to be DNA sequence-dependent.     

Non-invasive prenatal diagnosis by massively parallel sequencing of maternal plasma DNA

The presence of foetal DNA in the plasma of pregnant women has opened up new possibilities for non-invasive prenatal diagnosis. The use of circulating foetal DNA for the non-invasive prenatal detection of foetal chromosomal aneuploidies is challenging as foetal DNA represents a minor fraction of maternal plasma DNA. In 2007, it was shown that single molecule counting methods would allow the detection of the presence of a trisomic foetus, as long as enough molecules were counted. With the advent of massively parallel sequencing, millions or billions of DNA molecules can be readily counted. Using massively parallel sequencing, foetal trisomies 21, 13 and 18 have been detected from maternal plasma. Recently, large-scale clinical studies have validated the robustness of this approach for the prenatal detection of foetal chromosomal aneuploidies. A proof-of-concept study has also shown that a genome-wide genetic and mutational map of a foetus can be constructed from the maternal plasma DNA sequencing data. These developments suggest that the analysis of foetal DNA in maternal plasma would play an increasingly important role in future obstetrics practice. It is thus a priority that the ethical, social and legal issues regarding this technology be systematically studied.     

Manipulating the Plasmodium genome

Genome manipulation, the primary tool for assigning function to sequence, will be essential for understanding Plasmodium biology and malaria pathogenesis in molecular terms. The first success in transfecting Plasmodium was reported almost ten years ago. Gene-targeting studies have since flourished, as Plasmodium is haploid and integrates DNA only by homologous recombination. These studies have shed new light on the function of many proteins, including vaccine candidates and drug resistance factors. However, many essential proteins, including those involved in parasite invasion of erythrocytes, cannot be characterized in the absence of conditional mutagenesis. Proteins also cannot be identified on a functional basis as random DNA integration has not been achieved. We overview here the ways in which the Plasmodium genome can be manipulated. We also point to the tools that should be established if our goal is to address parasite infectivity in a systematic way and to conduct refined structure-function analysis of selected products.     

Detection of single DNA molecules by multicolor quantum-dot end-labeling

Observation of DNA–protein interactions by single molecule fluorescence microscopy is usually performed by using fluorescent DNA binding agents. However, such dyes have been shown to induce cleavage of the DNA molecule and perturb its interactions with proteins. A new method for the detection of surface-attached DNA molecules by fluorescence microscopy is introduced in this paper. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both extremities of a DNA molecule via sequence-specific hybridization and ligation. After the modified DNA molecules have been stretched on a glass surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots (QD). We demonstrate that under carefully selected conditions, the position and orientation of individual DNA molecules can be inferred with good efficiency from the QD fluorescence signals alone. This is achieved by selecting QD pairs that have the distance and direction expected for the combed DNA molecules. Direct observation of single DNA molecules in the absence of DNA staining agent opens new possibilities in the fundamental study of DNA–protein interactions. This work also documents new possibilities regarding the use of QD for nucleic acid detection and analysis.     

The Replication of DNA: II. The Number of Polynucleotide Strands in the Conserved Unit of DNA

The kinetics of degradation of DNA by deoxyribonuclease II have been studied, using the techniques of light scattering, viscosity, and titration. Theoretical equations have been derived for both random and non-random attacks, and all assumptions have been evaluated. It has been shown that these equations permit a valid calculation of the number of polynucleotide strands per molecule. The results have been verified by two independent experimental methods. DNA from proliferating sources was found to be four-stranded; DNA from non-proliferating sources was found to be two-stranded. The implications of these findings are discussed.     

Effect of supercoiling on the melting characteristics of heteropolynucleotides

Considering a supercoiled DNA molecule, having equal numbers of two distinct types of base-pairs, it has been shown theoretically that even for the extreme cases of mixing of the two types of base-pairs in a supercoiled DNA, the melting temperatures as well as the melting curves do not differ significantly. This indicates that these properties are practically independent of the detailed base sequence when the molecule is a covalently closed one and may be replaced by an equivalent homopolynucleotide whose binding energy is equal to the average base-pairing energy of the original DNA. This conclusion has been further supported by comparing the theoretical results with those obtained experimentally in the cases of polyoma DNA and phi X174 DNA. Finally, the effects of supercoiling on the cooperativity of melting and a few aspects of the differential melting characteristics of a supercoiled DNA have been discussed which provide a clear physical understanding of the process.     

Simian virus 40 DNA replication: characterization of gaps in the termination region.

A class of precursor DNA (pDNA) II molecules has been identified as the immediate precursor of simian virus 40 DNA I. A pDNA II molecule contains a strand of newly synthesized DNA with an interruption located in the region where DNA synthesis terminates (4). These pDNA II molecules have been isolated and further characterized. They are converted to covalently closed structures (simian virus 40 DNA I) only when they are treated in vitro with both T4 DNA polymerase and Escherichia coli ligase. After in vitro repair of pDNA II with T4 DNA polymerase and nucleoside triphosphates, approximately 7 mol of alpha-[32P]dATP is incorporated per mol of DNA II. Alkaline sucrose analysis of these gap-filled molecules, after they have been cleaved with Eco RI restriction endonuclease, has demonstrated that gaps are specifically located in the termination region. alpha-[32P]dATP is incorporated equally into the two labeled products that are generated by RI cleavage of these molecules. This indicates the presence of gaps in both the newly synthesized plus the minus strands. Electrophoretic analysis of the gap-filled molecules, after they have been cleaved with endonuclease Hind, has shown that gaps are localized in Hind fragments G and B and to a minor degree in fragment J. pDNA II molecules have the following properties. There is a gap in the newly synthesized linear DNA strand contained in the pDNA II molecule. Nicked pDNA II molecules cannot be detected. The two molecules that arise by segregation contain gaps in both of the complementary strands. Based on the amount of alpha-[32P]dATP incorporated and the rate of exonuclease III digestion of gap-filled molecules, it is estimated that the size of the gaps is between 22 and 73 nucleotides. Models for termination of DNA synthesis are proposed based on these findings.     

Plasmodium yoelii: a differential fluorescent technique using Acridine Orange to identify infected erythrocytes and reticulocytes in Duffy knockout mouse

Both human malarial parasite Plasmodium vivax and mouse malaria parasite Plasmodium yoelii use Duffy protein as the receptor for invasion and they preferentially invade reticulocytes. Recently, it has been shown that P. yoelii invades mouse reticulocytes by a Duffy independent pathway. Parasite invasion is generally visualized by time consuming staining procedures with dyes like Giemsa or Wright-Giemsa. Fluorochromatic dye like Acridine Orange has been used for instantaneous detection of parasites in RBCs. Acridine Orange binds to both DNA and RNA but with different emission spectra; and the binding can be distinguished with a fluorescent microscope using a green or a red filter, respectively. We have used this differential emission of Acridine Orange to determine P. yoelii invasion into erythrocytes and reticulocytes of Duffy positive and Duffy knockout mice. Moreover, we show that this method can be used to determine the maturity of reticulocytes in the peripheral blood of anemic mice.     

Polymorphism of the TRAP gene of Plasmodium falciparum

Natural sequence variation of the thrombospondin related anonymous protein (TRAP) gene of Plasmodium falciparum has been investigated by DNA analysis following the polymerase chain reaction amplification, and this shows the gene to be highly polymorphic. The region containing the sequence motif Trp-Ser-Pro-Cys-Ser-Val-Thr-Cys-Gly (WSPCSVTCG), common to TRAP, the circumsporozoite protein, properdin, and thrombospondin, was invariant. Elsewhere in the molecule, over 50 amino acid substitutions are described including the insertion of an in-frame, small-variable tandemly repeating motif between amino acid residues 352 and 353. Only one silent mutation was observed. Most nucleotide changes that occur in the first two codon positions result in conservative amino acid changes. Restriction fragment length polymorphism (RFLP) analysis was used to examine inheritance of TRAP in a cross between the HB3 and 3D7 clones of P. falciparum. Out of nine progeny examined, four possessed the HB3 gene and five the 3D7 gene. The TRAP gene hybridized to chromosome 13. Previous work has shown that a subtelomeric region of chromosome 13 from the 3D7 parent (marked by the HRP-III gene) was favoured strongly in this cross. The TRAP gene, however, is over 1 Mb away from this subtelomeric region and exhibits no such linkage because of chromosome crossovers. Five geographically separate isolates shared the same TRAP sequence as well as the same variant of the Th2R/Th3R region from the circumsporozoite protein. The correlation between independent markers in these isolates suggests that they have a common provenance.     

The effect of spermine on acid-base equilibrium in DNA molecule

The influence of spermine (Sp) on the acid-induced predenaturational and denaturational transitions in the DNA molecule structure has been studied by means of circular dichroism, spectrophotometric and viscometric titration at supporting electrolyte concentration 10 mM NaCl. The data available indicate that at [N]/[P] less than or equal to 0.60 (here [N] and [P] are molar concentrations of Sp nitrogen and DNA phosphours, respectively) the cooperative structural B----B(+)----S transitions are accompanied by the DNA double-helice winding. No competition for proton acceptor sites in the DNA molecule between H+ and Sp4+ cations has been observed when binding to neutral macromolecule. At 0.60 less than or equal to [N]/[P] less than or equal to 0.75 the displacement of the B----B(+)----S transitions midpoints to acidic pH region has been established. This is accompanied by DNA condensation and the appearance of differential scattering of circularly polarized light. The calculations carried out in the framework of the two-variable Manning theory have shown that the acid-induced reduction of the effective polyion charge density facilitates the Sp-induced DNA condensation. It has been shown that the acid-base equilibrium in the DNA molecule is determined by local [H+] in the 2-3 A hydrated monolayer of the macromolecule. An adequate estimation of [H+] can be obtained on the basis of the Poisson-Boltzman approach. The data obtained are consistent with recently proposed hypothesis of polyelectrolyte invariance of the acid-base equilibrium in the DNA molecule.     

The DNA untwisting enzyme from Saccharomyces cerevisiae. Partial purification and characterization.

The DNA untwisting enzyme has been partially purified from Saccharomyces cerevisiae. The enzyme exhibits a pH optimum of 7.3 to 7.6 in phosphate buffer, appears to require 0.15 M KCl for activity as determined by a DNA filter-binding assay, and is inhibited by N-ethylmaleimide. Like the untwisting enzymes from other eucaryotic cells, it can remove both positive and negative superhelical turns. A DNA molecule containing a single strand break was shown to be an intermediate in the untwisting reaction. Thermal stabilities of the enzyme from selected conditional lethal mutants defective in DNA synthesis have been examined and were found to be indistinguishable from the wild type enzyme.     

UV—A区段紫外线照射对DNA影响的拉曼光谱分析

本文检测了鲱鱼精DNA水溶液经不同时间UV－A紫外辐射后的拉曼光谱,研究结果表明,该区段紫外辐射比用UV－A和UV－B共同照射对DNA的影响要小,主链构象基本稳定。但经较长时间辐射仍会对鲱鱼精DNA造成损伤,受影响的部位主要是脱氧核糖和胸腺嘧啶碱基部分,UV－A对脱氧核糖的影响与UV－A加UV－B共同照射的结果作比较后,可以说明UV－A对脱氧核糖的损伤有累积的效应,而对于胸腺嘧啶的影响,从其各个指标的分析来看,有损伤但程度较小。本实验说明UV－A辐射条件下没有嘧啶二聚体的形成,也不存在6,4光产物形成的证明,但对于Dewar异构体的形成,有部分证明,与Taylor(1994)报道的结果相一致,UV－A没有造成DNA单链断裂现象。     

Nucleotide sequence-directed mapping of the nucleosomes of SV40 chromatin

In our previous work we have shown by comparison of experimental and computational data that the positions of the histone octamers bound to the DNA molecule appear to be completely sequence-dependent. This provides a convenient and quick method for locating the nucleosomes along the DNA molecule, as soon as the nucleotide sequence is known. Using this computational approach, the complete nucleosomal map of the SV40 minichromosome has been constructed. The map consists of 25 nucleosomes, with their coordinates (centers) being specified with high accuracy. The map is found to be in remarkable agreement with available experimental data.     

Nucleotide sequence-directed mapping of the nucleosomes of SV40 chromatin

In our previous work we have shown by comparison of experimental and computational data that the positions of the histone octamers bound to the DNA molecule appear to be completely sequence-dependent. This provides a convenient and quick method for locating the nucleosomes along the DNA molecule, as soon as the nucleotide sequence is known. Using this computational approach, the complete nucleosomal map of the SV40 minichromosome has been constructed. The map consists of 25 nucleosomes, with their coordinates (centers) being specified with high accuracy. The map is found to be in remarkable agreement with available experimental data.     

Dynamics of charge carrier transfer in a DNA molecule

An equivalent electric circuit has been developed which describes the charge transfer in DNA molecule. A computer simulation of the charge carrier transfer dynamics in the molecule has been performed based on this circuit. It was found that the switching time of a molecular junction lies in the femtosecond range and depends on the frequency of the input electric signal. An increase in the frequency of the input signal in the range from 1 GHz to 4 THz and a reduction of temperature lead to a decrease in the current passing through the DNA molecule. It has been shown that the sequence of the DNA base pairs defines the rate of localization and delocalization of holes and controls the signal propagation rate in the DNA molecule.     

Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments

The breakthroughs in single molecule spectroscopy of the last decade and the recent advances in super resolution microscopy have boosted the popularity of cyanine dyes in biophysical research. These applications have motivated the investigation of the reactions and relaxation processes that cyanines undergo in their electronically excited states. Studies show that the triplet state is a key intermediate in the photochemical reactions that limit the photostability of cyanine dyes. The removal of oxygen greatly reduces photobleaching, but induces rapid intensity fluctuations (blinking). The existence of non-fluorescent states lasting from milliseconds to seconds was early identified as a limitation in single-molecule spectroscopy and a potential source of artifacts. Recent studies demonstrate that a combination of oxidizing and reducing agents is the most efficient way of guaranteeing that the ground state is recovered rapidly and efficiently. Thiol-containing reducing agents have been identified as the source of long-lived dark states in some cyanines that can be photochemically switched back to the emissive state. The mechanism of this process is the reversible addition of the thiol-containing compound to a double bond in the polymethine chain resulting in a non-fluorescent molecule. This process can be reverted by irradiation at shorter wavelengths. Another mechanism that leads to non-fluorescent states in cyanine dyes is cis-trans isomerization from the singlet-excited state. This process, which competes with fluorescence, involves the rotation of one-half of the molecule with respect to the other with an efficiency that depends strongly on steric effects. The efficiency of fluorescence of most cyanine dyes has been shown to depend dramatically on their molecular environment within the biomolecule. For example, the fluorescence quantum yield of Cy3 linked covalently to DNA depends on the type of linkage used for attachment, DNA sequence and secondary structure. Cyanines linked to the DNA termini have been shown to be mostly stacked at the end of the helix, while cyanines linked to the DNA internally are believed to partially bind to the minor or major grooves. These interactions not only affect the photophysical properties of the probes but also create a large uncertainty in their orientation.