The Bombyx mori genome: analysis by DNA reassociation kinetics

The size and nucleotide sequence complexity of the Bombyx mori genome has been determined from the kinetics of reassociation of its DNA. Nonrepeated DNA comprises 55% of the genome, and the remainder is divided equally between sequences repeated roughly 500 and 50000 times. Non-repeated sequence DNA virtually free of repeated sequences was prepared by partial reassociation and subsequent fractionation on hydroxyapatite. The nucleotide sequence complexity of this component was determined relative to DNA from B. subtilis and E. coli. After correction for the size of the repeated sequence fraction, the DNA content of the Bombyx mori genome was calculated to be 0.53±0.02×10^?12 g. This value compares favorably with the DNA content of haploid B. mori spermatids and mature sperm determined cytophotometrically by Rasch (1973).     

Oscheius tipulae, a widespread hermaphroditic soil nematode, displays a higher genetic diversity and geographical structure than Caenorhabditis elegans

The nematode Oscheius tipulae belongs to the same family (Rhabditidae) as the model species Caenorhabditis elegans . Both species reproduce through self-fertilizing hermaphrodites and facultative males. Recent studies have shown that the self-fertile C. elegans and C. briggsae displayed a 20-fold lower genetic diversity than the male–female species C. remanei . Several explanations have been put forward to account for this difference, including their mode of reproduction and dynamic population structure. Here, we present the results of extensive worldwide sampling of O. tipulae , which we previously used as a laboratory organism for developmental genetics. We found that O. tipulae is much more widespread and common in soil throughout the world than Caenorhabditis species. We analysed 63 O. tipulae isolates from several continents using amplified fragment length polymorphism (AFLP). We found that O. tipulae harbours a 5-fold higher genetic diversity than C. elegans and C. briggsae . As in C. elegans , a high proportion of this diversity was found locally. Yet, we detected significant geographical differentiation, both at the worldwide scale with a latitudinal structure and between three localities in France. In summary, O. tipulae exhibited significantly higher levels of genetic diversity and large-scale geographical structure than C. elegans , despite their shared mode of reproduction. This species difference in genetic diversity may be explained by a number of other differences, such as population size, distribution, migration and dynamics. Due to its widespread occurrence and relatively high genetic diversity, O. tipulae may be a promising study species for evolutionary studies.     

The influence of fluorescent dye structure on the electrophoretic mobility of end-labeled DNA.

Over the past 10 years, fluorescent end-labeling of DNA fragments has evolved into the preferred method of DNA detection for a wide variety of applications, including DNA sequencing and PCR fragment analysis. One of the advantages inherent in fluorescent detection methods is the ability to perform multi-color analyses. Unfortunately, labeling DNA fragments with different fluorescent tags generally induces disparate relative electrophoretic mobilities for the fragments. Mobility-shift corrections must therefore be applied to the electrophoretic data to compensate for these effects. These corrections may lead to increased errors in the estimation of DNA fragment sizes and reduced confidence in DNA sequence information. Here, we present a systematic study of the relationship between dye structure and the resultant electrophoretic mobility of end-labeled DNA fragments. We have used a cyanine dye family as a paradigm and high-resolution capillary array electrophoresis (CAE) as the instrumentation platform. Our goals are to develop a general understanding of the effects of dyes on DNA electrophoretic mobility and to synthesize a family of DNA end-labels that impart identically matched mobility influences on DNA fragments. Such matched sets could be used in DNA sequencing and fragment sizing applications on capillary electrophoresis instrumentation.     

DNA isolated from Mycobacterium leprae: genome size, base ratio, and homology with other related bacteria as determined by optical DNA-DNA reassociation

DNA derived from Mycobacterium leprae (grown in armadillos) was isolated, purified, and analyzed spectrophotometrically. The genome size and the guanine-plus-cytosine content of M. leprae were 1.3 x 10(9) and 55.8%, respectively. Among selected strains of mycobacterial, nocardial, and corynebacterial species, Corynebacterium sp. 2628 LB, isolated from a human leprosy patient, showed the highest DNA homology with M. leprae. Of the DNAs derived from mycobacteria, those of M. tuberculosis and M. scrofulaceum showed a comparatively high reassociation with the DNMA of M. liprae.     

Genome analysis of ground squirrels of the genus Citellus (Rodentia,Sciuridae) I. DNA reassociation kinetics and genome size of eight species

Kinetic of reassociation of short DNA fragments were measured in eight ground squirrel species: Citellus undulatus, C. parryi, C. relictus, C. dauricus, C. citellus, C. pygmaeus, C. fulvus and C. major. It was shown that 30–50% of their genome were represented by repeated sequences forming three kinetic fractions, i.e., very fast (Cot<10^-3), fast (Cot 10^-3–3×10^-1) and intermediate (Cot 6×10^-1–6×10¹). Based on parameters of DNA reassociation kinetics genome sizes of Citellus were estimated to range from 2.7 pg (C. dauricus) to 3.9 pg (C. pygmaeus and C. fulvus). Variation in genome sizes involves both the repeated and the non-repeated sequence components to approximately equal extents in all the species except C. dauricus. The linear quantitative relation between C-banding heterochromatin and both very fast and fast reassociated DNA fractions was established, but no connection with the intermediate fraction was found. No distinet relation was revealed between parameters of DNA reassociation kinetics and taxonomic status of species within genus or with the chromosome number of the karyotype.     

DNA reassociation kinetics and genome complexity of a fish (Psetta maxima: Teleostei) and its gut parasite (Bothriocephalus gregarius: Cestoda).

1. The genomic structure of a fish (Psetta maxima) and of a Tapeworm (Bothriocephalus), who form a close host-parasite association, was determined by reassociation kinetics experiments. 2. Spectrophotometric readings of single-stranded versus double-stranded DNA separated on hydroxylapatite columns after reassociation at Cot values ranging from 0.0001 to 10(5) allowed the drawing of the reassociation curves of both genomes. 3. Different fractions according to their degree of repetitivity were evidenced, and the relative amounts of repetitive versus single-copy sequences, as well as their complexity, were calculated. 4. It appears that the amount of non-repetitive DNA is lower in the Tapeworm than in its vertebrate host, although the complexity of these single-copy sequences is the same.     

The Aedes aegypti genome: complexity and organization.

We describe the use of DNA reassociation kinetics to determine the total genome size and complexity together with the individual complexity and copy number of the single copy, middle repetitive and highly repeated DNA fractions of cell line and larval DNA from the mosquito, Aedes aegypti. The genome of Ae. aegypti is both large and complex, being one third the size of the human genome, and exhibits a short period interspersed repeat pattern. The implications of patterns of sequence arrangement and genome complexities for experiments aimed at isolating specific classes of DNA sequences, such as mobile genetic elements, are discussed.     

Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1

We used a fluorometric method to examine amyloid fibrils, in vitro. These fibrils in the case of both murine senile and secondary amyloidosis were purified to apparent homogeneity from the water-suspended fraction of the liver of senescence-accelerated mouse, using sucrose density ultracentrifugation, and then the following assays were performed. In the absence of amyloid fibrils, thioflavine T fluoresced faintly at the excitation and emission maxima of 350 and 438 nm, respectively. In the presence of amyloid fibrils, thioflavine T fluoresced brightly at the excitation and emission maxima of 450 and 482 nm, respectively, and the fluorescence change was linear from 0 to 2.0 micrograms/ml amyloid fibrils. This fluorescence was maximal around pH 9.0. Fluorescence intensity in the presence of a constant amount of amyloid fibrils reached a plateau with increase in the thioflavine T concentration. Normal high density lipoproteins which contain apo A-II, the precursor of amyloid fibrils in murine senile amyloidosis, and acute phase high density lipoproteins which contain serum amyloid protein A, the precursor of amyloid fibrils in secondary amyloidosis, showed little fluorescence. The fluorescence was considerably diminished when structure of the amyloid fibrils was disrupted by guanidine-HCl treatment. This method will be useful for the determination of amyloid fibrils in vitro.     

The interactions between the fluorescent dye thiazole orange and DNA

The interaction of the fluorescent dye thiazole orange (TO) with nucleic acids is characterized. It is found that TO binds with highest affinity to double-stranded (ds) DNA [log(K) ≈ 5.5 at 100 mM salt], about 5–10 times weaker to single-stranded polypurines, and further 10–1000 times weaker to single-stranded polypyrimidines. TO binds as a monomer to dsDNAs and poly(dA), both as a monomer and as a dimer to poly(dG) and mainly as a dimer to poly(dC) and poly(dT). The fluorescence quantum yield of TO free in solution is about 2 · 10⁻⁴, and it increases to about 0.1 when bound to dsDNA or to poly(dA), and to about 0.4 when bound to poly(dG). Estimated quantum yields of TO bound to poly(dC) and poly(dT) are about 0.06 and 0.01, respectively. The quantum yield of bound TO depends on temperature and decreases about threefold between 5 and 50°C. © 1998 John Wiley & Sons, Inc. Biopoly 46: 39–51, 1998     

Method for determination of nucleotide sequence homology between viral genomes by DNA reassociation kinetics.

A model and appropriate equations were derived for the quantitative estimation of nucleotide sequence homology between two partially related viral genomes by measurement of the initial rate of reassociation of one labeled DNA in the presence of a second unlabeled DNA. The validity and usefulness of this procedure were demonstrated by the analysis of the reassociation kinetics of labeled adenovirus 7 DNA in the presence of unlabeled adenovirus 2 DNA. Based on DNA reassociation, the extent of homology between adenovirus 2 and 7 genomes was found to be 10 to 12%. The duplex formed between adenovirus 2 and 7 DNA had the appropriate thermal stability for a well-matched DNA-DNA hybrid.     

Bacterial species determination from DNA-DNA hybridization by using genome fragments and DNA microarrays

Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays. Genomes from 12 well-characterized fluorescent Pseudomonas strains were labeled with Cy dyes and hybridized to the arrays. Cluster analysis of the hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria. Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity. In addition, the method does not require laborious cross-hybridizations and can provide an open database of hybridization profiles, avoiding the limitations of traditional DNA-DNA hybridization.     

The genome of alpha-proteobacteria : complexity, reduction, diversity and fluidity

The alpha-proteobacteria displayed diverse and often unconventional life-styles. In particular, they keep close relationships with the eucaryotic cell. Their genomic organization is often atypical. Indeed, complex genomes, with two or more chromosomes that could be linear and sometimes associated with plasmids larger than one megabase, have been described. Moreover, polymorphism in genome size and topology as well as in replicon number was observed among very related bacteria, even in a same species. Alpha-proteobacteria provide a good model to study the reductive evolution, the role and origin of multiple chromosomes, and the genomic fluidity. The amount of new data harvested in the last decade should lead us to better understand emergence of bacterial life-styles and to build the conceptual basis to improve the definition of the bacterial species.     

Catalysis of DNA reassociation by the Escherichia coli DNA binding protein: A polyamine-dependent reaction.

The Escherichia coli DNA binding protein, which binds co-operatively to single-stranded DNA, has been found to catalyse the formation of the DNA double helix from complementary strands in specified conditions. These conditions are different from the ones in which Alberts &; Frey (1970) found catalysis of DNA reassociation by the binding protein coded by gene 32 of phage T4. Although a 300-fold catalysis is observed in 10 mm-Mg²⁺ at pH 5·5, the catalytic efficiency of the binding protein drops sharply above pH 6 and is negligible at pH 7. Substitution of Ca²⁺ for Mg²⁺ extends slightly the pH range of strong catalysis up to pH 6·4, but catalysis again is slight at pH 7. When only Na⁺ or K⁺ is present, no catalysis is observed.At pH 7 catalysis appears to be a polyamine-dependent reaction: at 2 mm-spermidine a 5000-fold catalysis is found over a broad pH range, and spermine gives even higher rates. The mechanism of catalysis has not yet been studied in detail, but four properties of the reaction are noted here. (1) The DNA reassociation reaction follows apparent second-order kinetics. (2) For effective catalysis, the DNA binding protein must be in excess. (3) The catalytic efficiency increases strongly with DNA length. (4) The complex dependence of catalysis on the type of counterion and on pH suggests that other factors are involved in addition to melting out hairpin helices in the DNA single strands.The effect of the DNA binding protein on the rate of joining of the bacteriophage λ cohesive ends has also been studied, using a gel electrophoresis assay, for the joining of the EcoRI restriction fragments from the left and right hand ends of λ DNA. No catalysis of this joining reaction has been found.     

The size and structure of the DNA genome of symbiont xenosome particles in the ciliate Parauronema acutum

The size and structure of the DNA genome of xenosomes, bacterial endosymbionts of the marine hymenostome ciliate, Parauronema acutum 110-3, were investigated. Renaturation kinetic measurements, determined optically and by hydroxyapatite chromatography, suggested a genome size of 0.34 x 10(9) daltons. Sedimentation rate measurements of DNA gently released from the symbionts yielded molecules of comparable size. The analytical complexity, determined chemically, was 3.03 x 10(9) daltons. Consistent with these and other data is a model for the structure of the symbiont genome in which the DNA exists in the form of nine circularly permuted, double-stranded DNA molecules of unique sequence, each of molecular weight 0.34 x 10(9). It is suggested that xenosomes and certain symbionts found in ciliated protozoa may be extant forms of once free-living bacteria that have adapted to the intracellular environment.     

Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay.

We have developed a new assay to characterize the double-stranded DNA (dsDNA) binding properties of RecA protein. This assay is based on measurement of changes in the fluorescence of a 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complex upon RecA protein binding. The binding of RecA protein to a complex of DAPI and dsDNA results in displacement of the bound DAPI, producing a decrease in the observed fluorescence. DAPI displacement is dependent on both RecA protein and ATP; dATP and, to a lesser extent, UTP and dCTP also support the DAPI displacement reaction, but dGTP, GTP, dITP and TTP do not. Binding stoichiometry for the RecA protein-dsDNA complex measured by DAPI displacement is 3 bp per RecA protein monomer in the presence of ATP. These results, taken together with data for mutant RecA proteins, suggest that this DAPI displacement assay monitors formation of the high affinity DNA binding state of RecA protein. Since this state of RecA protein defines the form of the nucleoprotein filament that is active in DNA strand exchange, these findings raise the possibility that the RecA protein-dsDNA filament may possess a homologous pairing capacity.