Evolution of genome size and DNA base composition in reptiles

The evolution of genome size and base composition of DNA from various reptiles has been studied. DNA amount was measured cytophotometrically and GC concentration estimated by thermal denaturation. The Reptilia appear to be a fairly homogeneous group with respect to DNA quantity, although chelonians stand out because of their higher inter- and intrafamilial variability and DNA content. Quantitative DNA variations do not show a single evolutionary trend, but rather seem to have followed different patterns within each group.The differences in genome size between related species seem to be mainly the result of duplication or loss of DNA sequences characterized by a similar mean denaturation temperature. This agrees with observations of other authors that quantitative variations in reptiles are mainly due to differences in the amount of repetitive DNA.Several hypotheses on the significance of quantitative DNA variations in reptiles are discussed.     

Genome size and complexity of the obligate fungal pathogen,Bremia lactucae

The size and organization of the genome of Bremia lactucae, a highly specialized fungal pathogen of lettuce, has been characterized using dot blot genomic reconstructions, reverse genomic blots, and genomic DNA reassociation kinetics. The haploid genome contains 5 × 10⁷ bp of DNA and 65% of the nuclear DNA is repeated. Low copy sequences are interspersed with repeated sequences in a short-period interspersion pattern. This pattern of genome organization is different to that described for other fungi. Although most fungi have been shown to contain some form of repetitive DNA other than the ribosomal repeat, the high percentage of repetitive DNA and the interspersion of low copy and repeated sequences are atypical of fungi characterized previously.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.     

Reassociation Kinetics and Cytophotometric Characterization of Peanut (Arachis hypogaea L.) DNA

The base composition of peanut (var. NC-17) DNA determined from thermal denaturation profiles showed an average guanine plus cystosine content of 34% which was in close approximation to 36% guanine plus cytosine calculated from the buoyant density. Buoyant density also indicated the absence of satellite DNA. The genome size, 2.0 × 10⁹ base pairs, as determined by reassociation kinetics of the single copy DNA was close to the genome size determined by cytophotometry, 2.1 × 10⁹ base pairs. Peanut DNA averaging 450 to 600 base pairs long, reassociated in phosphate buffer and fractionated by hydroxylapatite, indicated a DNA genome composition of 36% nonrepetitive or single copy DNA; reassociation in formamide and followed by optical methods indicated the repetitive DNA possesses highly repeated, intermediately repeated and rarely repeated components of DNA with DNA sequences repeated on the average about 38,000, 6,700, and 200 times each. Different criteria of reassociation in formamide revealed further subdivisions of these four separate components of DNA. The DNA of above mentioned NC-17 variety compared to Florigiant variety showed no differences in thermal denaturation profiles, buoyant density, or in genome size.     

Cloning and characterization ofDaphnia mitochondrial DNA

Summary The mitochondrial genome ofDaphnia pulex (Crustacea, Cladocera) was cloned as a single fragment into the plasmid vector pUC12. The genome size, estimated from restriction endonuclease fragment lengths, is 15,400±200 base pairs. The GC content, estimated from thermal denaturation studies, is 42%. The positions of 39 cleavage sites were mapped for 14 restriction enzymes. The distribution of these sites within the genome is random (P=0.44). Heterologous hybridizations withDrosophila sylvestris mitochondrial DNA (mtDNA) probes indicate that gene orders withinDaphnia andDrosophila mtDNAs are similar.     

Genome Size, Complexity, and Ploidy of the Pathogenic Fungus Histoplasma capsulatum

The genome size, complexity, and ploidy of the dimorphic pathogenic fungus Histoplasma capsulatum was determined by using DNA renaturation kinetics, genomic reconstruction, and flow cytometry. Nuclear DNA was isolated from two strains, G186AS and Downs, and analyzed by renaturation kinetics and genomic reconstruction with three putative single-copy genes (calmodulin, α-tubulin, and β-tubulin). G186AS was found to have a genome of approximately 2.3 × 10⁷ bp with less than 0.5% repetitive sequences. The Downs strain, however, was found to have a genome approximately 40% larger with more than 16 times more repetitive DNA. The Downs genome was determined to be 3.2 × 10⁷ bp with approximately 8% repetitive DNA. To determine ploidy, the DNA mass per cell measured by flow cytometry was compared with the 1n genome estimate to yield a DNA index (DNA per cell/1n genome size). Strain G186AS was found to have a DNA index of 0.96, and Downs had a DNA index of 0.94, indicating that both strains are haploid. Genomic reconstruction and Southern blot data obtained with α- and β-tubulin probes indicated that some genetic duplication has occurred in the Downs strain, which may be aneuploid or partially diploid.     

Examination of Genome Homogeneity in Prokaryotes Using Genomic Signatures

Background

DNA word frequencies, normalized for genomic AT content, are remarkably stable within prokaryotic genomes and are therefore said to reflect a “genomic signature.” The genomic signatures can be used to phylogenetically classify organisms from arbitrary sampled DNA. Genomic signatures can also be used to search for horizontally transferred DNA or DNA regions subjected to special selection forces. Thus, the stability of the genomic signature can be used as a measure of genomic homogeneity. The factors associated with the stability of the genomic signatures are not known, and this motivated us to investigate further. We analyzed the intra-genomic variance of genomic signatures based on AT content normalization (0^th order Markov model) as well as genomic signatures normalized by smaller DNA words (1^st and 2^nd order Markov models) for 636 sequenced prokaryotic genomes. Regression models were fitted, with intra-genomic signature variance as the response variable, to a set of factors representing genomic properties such as genomic AT content, genome size, habitat, phylum, oxygen requirement, optimal growth temperature and oligonucleotide usage variance (OUV, a measure of oligonucleotide usage bias), measured as the variance between genomic tetranucleotide frequencies and Markov chain approximated tetranucleotide frequencies, as predictors.

Principal Findings

Regression analysis revealed that OUV was the most important factor (p<0.001) determining intra-genomic homogeneity as measured using genomic signatures. This means that the less random the oligonucleotide usage is in the sense of higher OUV, the more homogeneous the genome is in terms of the genomic signature. The other factors influencing variance in the genomic signature (p<0.001) were genomic AT content, phylum and oxygen requirement.

Conclusions

Genomic homogeneity in prokaryotes is intimately linked to genomic GC content, oligonucleotide usage bias (OUV) and aerobiosis, while oligonucleotide usage bias (OUV) is associated with genomic GC content, aerobiosis and habitat.     

Whole-genome survey and phylogenetic analysis of Gadus macrocephalus

Gadus macrocephalus (Pacific cod) is an economically important species on the northern coast of the Pacific. Although numerous studies on G. macrocephalus exist, there are few reports on its genomic data. Here, we used whole-genome sequencing data to elucidate the genomic characteristics and phylogenetic relationship of G. macrocephalus. From the 19-mer frequency distribution, the genome size was estimated to be 658.22 Mb. The heterozygosity, repetitive sequence content and GC content were approximately 0.62%, 27.50% and 44.73%, respectively. The draft genome sequences were initially assembled, yielding a total of 500,760 scaffolds (N50 = 3565 bp). A total of 789,860 microsatellite motifs were identified from the genomic data, and dinucleotide repeat was the most dominant simple sequence repeat motif. As a byproduct of whole-genome sequencing, the mitochondrial genome was assembled to investigate the evolutionary relationships between G. macrocephalus and its relatives. On the basis of 13 protein-coding gene sequences of the mitochondrial genome of Gadidae species, the maximum likelihood phylogenetic tree showed that complicated relationships and divergence times among Gadidae species. Demographic history analysis revealed changes in the G. macrocephalus population during the Pleistocene by using the pairwise sequentially Markovian coalescent model. These findings supplement the genomic data of G. macrocephalus, and make a valuable contribution to the whole-genome studies on G. macrocephalus.     

Characterization of DNA from the BasidiomyceteSchizophyllum commune

We have examined the whole-cell DNA of homokaryons and dikaryons ofSchizophyllum commune by physical methods and reassociation kinetics. DNA with a single-strand length of 2800 bases was routinely isolated with urea—phosphate buffers and hydroxyapatite. Melting profiles indicate the bulk of the DNA to consist of a 57 ± 1.22% guanine plus cytosine content (GC) with a small amount of DNA melting at temperatures indicative of 22.1% GC. Analytical ultracentrifugations in CsCl gradients revealed a single band with buoyant density 1.718 g/ml equating to 59% GC. On the other hand, cesium chloride isopycnic centrifugation in the preparative ultracentrifuge disclosed a light satellite (ϱ = 1.687 g/ml, 27% GC) constituting about 2% of the whole-cell DNA. Analyses of the kinetics of reassociation of the whole-cell DNA by three different methods estimate the DNA of anS. commune homokaryotic cell to be 8.6 times that of anEscherichia coli cell (mean, 3.6 × 10⁷ nucleotide pairs, NTP; range of experimental values 3.1 − 4.1 × 10⁷ NTP). The dikaryotic cell would contain approximately twice this amount of DNA. We detect no other differences between the DNA of homokaryotic and dikaryotic cells as described within the limits of these methods. The reassociation data are best described with a single unique component accounting for 80–90% of the DNA and with a single repetitive fraction of 10 ± 2% of whole-cell DNA. Some ambiguity surrounds the repetition frequency and sequence complexity of the repetitive fraction because it does not reassociate well as a separate fraction (i.e., it does not reassociate with the anticipated rate constant, nor does it approach completion of the reaction) after being isolated by denaturation, reassociation, and isolation on hydroxyapatite.     

Classification of the Legionnaires' disease bacterium: An interim report

Deoxyribonucleic acid (DNA) from strains of the Legionnaires' disease bacterium (LDB) was characterized in order to aid in the proper classification of this organism. The genome size of LDB DNA was estimated at 2.5×10⁹ daltons by reassociation kinetics; a guanine-plus-cytosine content of LDB of 39% was established by optical thermal denaturation and buoyant density ultracentrifugation measurements. DNA relatedness studies on 12 strains of the LDB indicated that they were all members of the same species. DNA relatedness studies have thus far failed to show that the LDB is significantly related to any other organism, including all members of Enterobacteriaceae,Pasteurella multocida, Francisella tularensis, Rochalimaea quintana, Vibrio species,Staphylococcus epidermidis, andFlavobacterium meningosepticum.     

Nuclear DNA content of Pongamia pinnata L. and genome size stability of in vitro-regenerated plantlets

Pongamia pinnata L. is a multipurpose versatile legume that is well known as a prospective feedstock biodiesel species. However, to date, there has been little genomic research aimed at the exploitation of the biotechnological potential of this species. Genetic characterization of any plant is a challenging task when there is no information about the genome size and organization of the species. Therefore, the genome size of P. pinnata was estimated by flow cytometry with respect to two standards (Zea mays and Pisum sativum), and compared with that of in vitro-raised plants (nodal segment, in vitro-rooted plantlets and acclimatized in vitro plants) to study the potential effect of somaclonal variation on genome size. This method can be used to support the establishment of true-to-type plants to encourage afforestation programs. Modified propidium iodide/hypotonic citrate buffer was used for isolation of the intact nuclei. The 2C DNA value of this species was estimated to be 2.51?±?0.01 pg. Statistically, there was no significant difference in the DNA content of the in vitro-grown plants and mother plant at α?=?0.05. As a result of the low genome size of P. pinnata, a species that has adapted itself to a wide range of edaphic and ecological condition, we can now proceed for its next generation sequencing and genomic diversity studies.     

Genomic distribution of repeated and single copy DNA sequences in Penicillium funiculosum

Study of the reassociation kinetics of Penicillium funiculosum DNA (fragment size > 20 kb) revealed an interspersion of repeated and single-copy DNA sequences. The fragment length of interspersed single-copy DNA was estimated to be more than 9 kb from a curve relating the fraction of DNA binding to hydroxyapatite as a function of DNA fragment length. The length of interspersed short and long repeated nucleotide sequences was determined to be 0.55 kb and > 20 kb, respectively, by agarose gel electrophoresis. The genomic organization of P. funiculosum is thus significantly different from that in other fungi.     

Deoxyribonucleic acid base composition and genome size of Prochloron

The DNA base composition of the photosynthetic prokaryote Prochloron was determined (on samples collected from the natural environment) to be 40.8 mol% GC. The sharp differential melting curve indicated the absence of significant quantities of contaminating DNA from other organisms. The genome size, estimated from the renaturation kinetics of thermally denatured DNA, was 3.59×10⁹ daltons mol. wt, similar to that of many other prokaryotes. The fact that Prochloron has not yet been cultured in the laboratory cannot, therefore, be attributed to a reduced genetic information content.     

Size and complexity of the nuclear genome of the ectomycorrhizal fungus Paxillus involutus

The basidiomycete Paxillus involutus is forming ectomycorrhizal symbiosis with a broad range of forest trees. Reassociation kinetics on P. involutus nuclear DNA indicated a haploid genome size of 23 Mb including 11% of repetitive DNA. A similar genome size (20 Mb) was estimated by genomic reconstruction analysis using three single copy genes. To assess the gene density in the P. involutus genome, a cosmid containing a 33-kb fragment of genomic DNA was sequenced and used to identify putative open reading frames (ORFs). Twelve potential ORFs were predicted, eight displayed significant sequence similarities to known proteins found in other organisms and notably, several homologues to the Podospora anserina vegetative incompatibility protein (HetE1) were found. By extrapolation, we estimate the total number of genes in the P. involutus haploid genome to approximately 7700.     

Size and complexity of the nuclear genome of Colletotrichum graminicola.

DNA reassociation was used to estimate GC content, size, and complexity of the nuclear genomes of Colletotrichum from maize and sorghum. Melting-temperature analysis indicated that the GC content of the maize pathotype DNA was 51% and that the GC content of the sorghum pathotype was 52%. DNA reassociation kinetics employing S1 nuclease digestion and an appropriately modified second-order equation indicated that the genome sizes of the maize and sorghum pathotypes were 4.8 x 10(7) bp, and 5.0 x 10(7) bp, respectively. Genomic reconstruction experiments based on Southern blot hybridization between a cloned single-copy gene, PYR1 (orotate phosphoribosyl transferase), and maize-pathotype DNA confirmed the size of the nuclear genome. The single-copy component of the genomes of both pathotypes was estimated at about 90%. For both pathotypes, ca. 7% of the genome represented repetitive DNA, and 2 to 3% was foldback DNA.     

Elongation of repetitive DNA by DNA polymerase from a hyperthermophilic bacterium Thermus thermophilus

Short repetitive DNA sequences are believed to be one of the primordial genetic elements that served as a source of complex large DNA found in the genome of modern organisms. However, the mechanism of its expansion (increase in repeat number) during the course of evolution is unclear. We demonstrate that the DNA polymerase of the hyperthermophilic bacterium Thermus thermophilus can elongate oligoDNA with several tandem repeats to very long DNA in vitro. For instance, 48mer repetitive oligoDNA (TACATGTA)₆, which has 25% GC content and a palindromic sequence, can be elongated up to ~10 000 bases by DNA polymerase at 74°C without template DNA. OligoDNA having a different GC content or a quasi-palindromic sequence can also be elongated, but less efficiently. A spectroscopic thermal melting experiment with the oligoDNA showed that its hairpin–coil transition temperature was very close to the elongation reaction temperature (74°C), but was much higher than the temperature at which duplex oligoDNA can exist stably. Taken together, we conclude that repetitive oligoDNA with a palindromic or quasi-palindromic sequence is elongated extensively by a hyperthermophilic DNA polymerase through hairpin–coil transitions. We propose that such an elongation mechanism might have been a driving force to expand primordial short DNA.     

Isolation and characterization ofAnaplasma marginale DNA

Two procedures were used to isolateAnaplasma marginale bodies from bovine erythrocytes. DNA extracted from bodies prepared by the second method was free of any detectable bovine DNA contamination.Anaplasma marginale DNA was analyzed by agarose gel electrophoresis of endonuclease restriction fragments and by reassociation kinetics. Genome size was estimated to be 340 kb. Base composition of the DNA was 33 mol% guanine+ cytosine (G+C), determined from its thermal denaturation temperature.Anaplasma marginale has a very small genome compared with that of other bacteria and has a low G+C content. It is proposed thatA. marginale may be a close, but degenerate, relative of the rickettsiae.     

Nuclear DNA characterization of two species ofVicia:Vicia bithynica L. andVicia narbonensis L.

The speciesVicia bithynica andVicia narbonensis, from the same subgeneric section ofVicia faba, show variations in nuclear DNA content Nuclear DNAs, extracted from root tips of the twoVicia species, were characterized by thermal denaturation, analytical ultracentrifugation and reassociation kinetics. The thermal denaturations of DNA, the number of DNA components reassociating with second order kinetics, the proportion of repeated DNA sequences, the frequency of the repeated DNA classes are reported and compared to previous data onVicia faba DNA. Feulgen absorptions at different thresholds of optical density⁺ of interphase nuclei in cytological preparations of the root meristems ofV. bithynica andV. narbonensis are determined and compared withV. faba analogous determinations. The results, confirming that plant genome is highly flexible, are discussed in relation to other data on the interspecific variations of the nuclear DNA content.     

DNA Base composition and repetitious DNA in several conifers

Coniferous DNA was analyzed by ultraoentrifugation and thermal denaturation and renaturation to determine base composition and the presence of repetitive sequences. The percent quanine plus cytosine values among species were constant and independent of DNA quantity per cell, whereas the proportion of repetitive DNA per genome was greatest in those species that had the highest DNA content.     

Genome survey sequencing of purple elephant grass (<Emphasis Type="Italic">Pennisetum purpureum</Emphasis> Schum ‘Zise’) and identification of its SSR markers

Elephant grass (Pennisetum purpureum) is a perennial grass in the Poaceae family with high tolerance and one of the best forage plants. Despite its economic importance, the inheritance information of P. purpureum has remained largely unknown. To obtain the whole reference genome, we first conducted a genome survey of P. purpureum. Next-generation sequencing (NGS) was used to perform the de novo whole genome sequencing. As a result, the estimated genome size of elephant grass was 2.01 Gb, with 71.36% repetitive elements. The heterozygosity was 1.02%, which indicates a highly heterozygous genome. The retroelements (9.36%) were the most repetitive elements, followed by DNA transposons (3.66%). In the meantime, 83,706 high-quality genomic simple sequence repeat (SSR) markers, in which the greatest SSR unit length was 3, were developed. Thirty pairs of SSR markers were randomly selected to verify the efficiency and all of them yielded clear amplification products, among which 28 pairs (93.3%) of the primers showed polymorphism. The genome data obtained in this research provided a large amount of gene resources for further investigating Pennisetum species.