A thermal denaturation study of the genomic DNAs from the North American genera of the fish family Percidae

The genomic DNAs of 1 1 species of percid fishes representing the five recognized North American genera are characterized using data from thermal denaturation assays. Base compositions were estimated from the transitional melting temperature of native and sonicated DNA and expressed as per cent guanine-cytosine (%GC) values. Among genera, %GC values for native DNAs (c, 23,000 base pairs in length) range between 38.3% GC for yellow perch, Perca flavescens (Mitchill), to 43.2% GC for sauger, Stizostedion cunadense (Smith). Significant variation in %GC values was observed among surveyed genera of the subfamily Percinae, which include Perca, Percinu, Etheostoma and Ammocrypfa . Melting profiles were generated for each species, and distinct GC rich regions were identified within the genomes of walleye, Sfizostcdion virreum (Mitchill) and Etheostoma spp. Compositional heterogeneity (CH) and asymmetry values were calculated from melting profile data. Patterns of variation in genomic characters differed among the genera surveyed. Members of the speciose genus Etheostomu showed relatively little variation in genomic characters, whereas Stizosredion exhibited significant interspecific variation.     

Characteristics of nuclear DNA in the genus Oryza

Summary DNAs have been isolated from various Oryza species and studied using physical techniques. The percent of guanine plus cytosine has been determined by thermal denaturation. While the base composition varied between the species, no heterogeneity in the base pair distribution was observed. Renaturation kinetics data of DNAs from different species show that the proportion of repeated DNA sequences vary considerably depending on the DNA content per cell, whereas the nonrepetitive DNA component remains relatively constant. These results suggest that in addition to a small range of DNA variation between the species, changes in the base composition and proportion of repeated sequences have accompanied divergence of the species within the genus.     

Mechanisms of chromosome banding

Prior studies on subfractions of mouse and Kangaroo rat DNA have suggested that variations in base concentration within a given genome may not be great enough to account for Q-banding. To examine this with another species, calf DNA was subfractionated by CsCl ultracentrifugation into GC-rich satellites and the main band DNA was further fractionated into AT-rich, intermediate and GC-rich portions. The effect of varying concentrations of these DNAs on quinacrine and Hoechst 33258 fluorescence was examined. Although with both compounds there was less fluorescence in the presence of the GC-rich satellites than main band fractions, these results per se did not answer the question of whether the variation in base composition alone was adequate to account for chromosome banding. To answer this the fluorescence observed in the presence of DNA of a given base composition was related to the fluorescence observed in the presence of DNA of 40% GC content (F/F40). This allowed the derivation of a term B which indicated the relative change in fluorescence per 1% change in base composition of DNA. To determine the percent change in fluorescence observed in Q-banding, the photoelectric recordings of Caspersson et al. (1971) were used. From these data we conclude: 1. Quinacrine is twice as sensitive to changes in base composition as Hoechst 33258. 2. Variation in the base content of DNA along the chromosome is sufficient to account for most Q-banding, except possibly for some of the extremes of quinacrine fluorescence. This was further examined with daunomycin. Even though daunomycin gives good fluorescent banding, DNAs varying in base composition from 100 to 40% GC content all resulted in the same relative fluorescence of 0.03. However, in the presence of poly (dA-dT) the relative fluorescence was 0.85, indicating a great sensitivity to very AT-rich DNA. This suggests that with daunomycin and possibly other fluorochromes, stretches of very AT-rich DNA may be more important in fluorescent banding than simple variation in mean base composition.     

Genome size variation in North American minnows (Cyprinidae). II. Variation among 20 species

Genome sizes (nuclear DNA contents) from 200 individuals representing 20 species of North American cyprinid fishes (minnows) were examined spectrophotometrically. The distributions of DNA values of individuals within populations of the 20 species were essentially continuous and normal; the distribution of DNA values among species was continuous and overlapping. These observations suggest that changes in DNA quantity in cyprinids are small in amount, involve both gains and losses of DNA, and are cumulative and independent in effect. Significant heterogeneity in mean genome size occurs both between individuals within populations of species and among species. The former averages maximally around 6% of the cyprinid genome and is nearly the same as the amount of DNA theoretically needed for the entire cyprinid structural gene component. The majority of the DNA content variation among the 20 species is distributed above the level of individuals within populations. Comparisons of average genome size difference or distance between individuals drawn from different levels of taxonomic organization indicate that considerably greater divergence in genome size has occurred in the extremely speciose cyprinid genus Notropis as compared with other North American cyprinid genera. This may suggest that genome size change is concentrated in speciation episodes. Finally, no associations were found between interspecific variation in genome size and five life-history characters. This suggests that much of the variation in genome size within and among the 20 species may be phenotypically inconsequential.     

Characterization of a highly repeated satellite DNA from the cyprinid fish Notropis lutrensis

1. A highly repeated, satellite DNA family from the North American cyprinid fish, Notropis lutrensis, was identified as a fragment band following restriction endonuclease enzyme digestion and agarose gel electrophoresis of genomic DNA; evidence of a tandem arrangement of the satellite in the genome was demonstrated by the formation of "ladders" in partial restriction endonuclease digests. 2. The satellite family was estimated densitometrically to comprise 7-8% of the N. lutrensis genome; mapping experiments using isolated and purified monomer repeat units of the satellite uncovered nine sites for seven different restriction enzymes. 3. A monomeric repeat unit of the satellite was cloned and sequenced, and found to be 174 base pairs in length and to have a base composition of 47% G + C (guanine + cytosine); computer analysis of the sequence revealed 13 new restriction sites for 12 additional enzymes. 4. Computer analysis also revealed that a large degree of internal redundancy in the monomer unit exists in the form of both direct and inverted repeating units, and that the entire sequence, starting with one base in either orientation, constitutes an open reading frame. In all but the last characteristic, the N. lutrensis satellite DNA is very similar to satellite DNAs in other eukaryotes.     

Base- and sequence-dependent binding of aristololactam beta-D-glucoside to deoxyribonucleic acid

The dependence on base-pair composition and sequence specificity of the (aristololactam beta-D-glucoside)-DNA interaction was examined by spectrophotometric, spectrofluorometric, spectropolarimetric, thermal melting, thermodynamic, and viscometric studies. Binding of this alkaloid to various natural and synthetic DNAs was dependent upon the base composition and sequences of DNA. The binding parameters obtained from spectrophotometric analysis, according to an excluded-site model, indicated a relatively high affinity of the alkaloid binding to GC-rich DNA and alternating GC polymer. This affinity was further evidenced by the quenching of fluorescence intensity, decrease in quantum yield, and perturbations in circular dichroic spectrum. The alkaloid stabilized all DNAs against thermal denaturation. The temperature dependence of the binding constants was used to estimate the thermodynamic parameters involved in the complex formation of the alkaloid with various DNAs. The negative enthalpy and entropy change increased with increasing GC content of DNA and also compensated one another to produce a relatively small Gibbs free energy change. Viscometric studies showed that in the strong binding region the increase of contour length of DNA depended strongly on its base composition and sequence of bases, being larger for GC-rich DNA and alternating GC polymer. On the basis of these observations, it is concluded that the alkaloid binds to DNA by a mechanism of intercalation and exhibits considerable specificity toward alternating GC polymer.     

Homologies of repetitive DNA sequences among Crustacea

The interrelationships of a number of Crustacea were measured by nucleic acid hybridization techniques, with special emphas is on the question of whether GC-rich satellite DNA contains nucleotide sequences homologous to sequences found in other Crustacea with and without similar satellite DNAs. Repetitious sequences from both main-band DNA and GC-rich satellite DNA from the land crab, Gecarcinus lateralis, were hybridized to the total DNAs of crustaceans ranging from the brine shrimp (Subclass: Branchiopoda) to the North American lobster (Homarus americanus, Subclass: Malacostraca; Suborder: Repantia; Section: Macrura) and the true crabs (Subclass: Malacostraca; Suborder: Reptantia; Section: Brachyura). Approximately half of the Gecarcinus repetitious main-band DNA sequences were found to be represented in the DNA of the other true crabs, while a lesser but still significant amount of homology (5 to 10%) to the GC-rich satellite DNA was observed. We also observed a significant amount of homology of the Gecarcinus GC-rich satellite to other crustacean DNAs, even at the level of a different taxonomic Section. This is the first observation of hybrid formation between a purified satellite and DNAs from other organisms under stringent hybridization conditions.Research sponsored by the U.S. Atomic Energy Commission under contact with the Union Carbide Corporation.Research performed while an Oak Ridge Graduate Fellow under appointment from the Oak Ridge Associated Universities in partial fulfillment of the Ph. D. degree from the University of Tennessee, Knoxville, Tennessee.     

Amino Acid Usage Is Asymmetrically Biased in AT- and GC-Rich Microbial Genomes

Introduction

Genomic base composition ranges from less than 25% AT to more than 85% AT in prokaryotes. Since only a small fraction of prokaryotic genomes is not protein coding even a minor change in genomic base composition will induce profound protein changes. We examined how amino acid and codon frequencies were distributed in over 2000 microbial genomes and how these distributions were affected by base compositional changes. In addition, we wanted to know how genome-wide amino acid usage was biased in the different genomes and how changes to base composition and mutations affected this bias. To carry this out, we used a Generalized Additive Mixed-effects Model (GAMM) to explore non-linear associations and strong data dependences in closely related microbes; principal component analysis (PCA) was used to examine genomic amino acid- and codon frequencies, while the concept of relative entropy was used to analyze genomic mutation rates.

Results

We found that genomic amino acid frequencies carried a stronger phylogenetic signal than codon frequencies, but that this signal was weak compared to that of genomic %AT. Further, in contrast to codon usage bias (CUB), amino acid usage bias (AAUB) was differently distributed in AT- and GC-rich genomes in the sense that AT-rich genomes did not prefer specific amino acids over others to the same extent as GC-rich genomes. AAUB was also associated with relative entropy; genomes with low AAUB contained more random mutations as a consequence of relaxed purifying selection than genomes with higher AAUB.

Conclusion

Genomic base composition has a substantial effect on both amino acid- and codon frequencies in bacterial genomes. While phylogeny influenced amino acid usage more in GC-rich genomes, AT-content was driving amino acid usage in AT-rich genomes. We found the GAMM model to be an excellent tool to analyze the genomic data used in this study.     

Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence.

The vibrational spectra of four genomic and two synthetic DNAs, encompassing a wide range in base composition [poly(dA-dT). poly(dA-dT), 0% G + C; Clostridium perfringens DNA, 27% G + C; calf thymus DNA, 42% G + C; Escherichia coli DNA, 50% G + C; Micrococcus luteus DNA, 72% G + C; poly(dG-dC).poly(dG-dC), 100% G + C] (dA: deoxyadenosine; dG: deoxyguanosine; dC: deoxycytidine; dT: thymidine), have been analyzed using Raman difference methods of high sensitivity. The results show that the Raman signature of B DNA depends in detail upon both genomic base composition and sequence. Raman bands assigned to vibrational modes of the deoxyribose-phosphate backbone are among the most sensitive to base sequence, indicating that within the B family of conformations major differences occur in the backbone geometry of AT- and GC-rich domains. Raman bands assigned to in-plane vibrations of the purine and pyrimidine bases-particularly of A and T-exhibit large deviations from the patterns expected for random base distributions, establishing that Raman hypochromic effects in genomic DNA are also highly sequence dependent. The present study provides a basis for future use of Raman spectroscopy to analyze sequence-specific DNA-ligand interactions. The demonstration of sequence dependency in the Raman spectrum of genomic B DNA also implies the capability to distinguish genomic DNAs by means of their characteristic Raman signatures.     

Spectral analysis of high resolution direct-derivative melting curves of DNA for instantaneous and total base composition.

Derivative melting profiles of DNA have been obtained directly by recording the difference in absorbance between two identical solutions maintained at a small constant temperature differential. This deltaA is monitored continuously with increasing temperature in a ratio recording spectrophotometer. Resolution of complex hyperfine structure in the profiles of small homogeneous viral DNAs appears to be significantly better than has been produced by various numerical methods of differentiation. In addition, a spectral method has been modified that permits easy analysis for DNA base composition from the ratio of derivative melting curves obtained at 282 and 260 nm. Eight bacterial and three vertebrate DNAs have been analyzed for total base composition from the product of the instantaneous base composition at small temperature intervals (0.05 degrees C) throughout the entire melting region and the integrated area of the 282 nm profile. The results are in excellent agreement with values determined by traditional methods.     

真核生物DNA非编码区的组分分析

在全基因组水平上,用直方图、混沌表示灰度图、距离差异度和信息熵差异度四种方法,研究了拟南芥、线虫、果蝇的DNA内含子、基因间隔区DNA、外显子三种区域的核苷酸短序列组分及组分复杂度.结果表明：a.不同基因组之间,不管基因数目多少,用4种方法得到的外显子部分其组分复杂度都比较接近,而非编码区部分的组分复杂度却很大.这一点定量地说明了物种之间的复杂程度,主要不体现在编码区部分,而体现在非编码区部分.b.同一基因组中,内含子的核苷酸短序列组分复杂度都是相似的,外显子和intergenic DNA部分的组分复杂度也是相似的.c.内含子和intergenic DNA在转录、剪切、二级结构等方面有很大的不同,但它们在核苷酸短序列组分上的差异却很小,说明内含子和intergenic DNA在转录、剪切、二级结构上的不同并不通过核苷酸短序列组分来进行限制.     

Determination of Melting Sequences in DNA and DNA-Protein Complexes by Difference Spectra

A graphical formula is presented for determining the base ratio of melted DNA. By use of this formula, the composition of sequences which melt in different portions of the melting curves of Clostridium DNA, Escherichia coli DNA, and mouse DNA were determined. As the DNA melts, the per cent of adenine and thymine (AT) in the melted sequences decreases linearly with temperature. The average composition of sequences which melt in a given part of the melting curve is proportional to the base ratio of the DNA. The concentration and average composition of sequences were determined for three parts of the melting curves of the DNA samples, and a frequency distribution curve was constructed. The curve is symmetrical and has a maximum at about 56% AT. The distribution of GC-rich sequences on the E. coli chromosome was estimated by shearing, partially melting, and fractionating the DNA on hydroxylapatite. GC-rich sequences appear to occur every thousand base pairs, and have a maximum length of about 180 base pairs. The graphical formula was applied to the determination of the composition of sequences which melt in different parts of the melting curve of chromatin. Throughout the melting curve, the composition of the melting sequences is about 60% AT, which appears to suggest that relatively long sequences are melting simultaneously. Their melting temperature may be a function of the composition of the protein on different parts of the DNA. The problem of light scattering in DNA-protein and DNA was also investigated. A formula is presented which corrects for light scattering by relating the intensity of the scattered light to the rate of change of absorbance of DNA with wavelength.     

Liverwort genomes display extensive structural variations

PIKE, L. M., HU, A., RENZAGLIA, K. S. & MUSICH, P. R., 1992. Liverwort genomes display extensive structural variations. Analyses of the total genomic DNA of eight species of liverworts and two species of green algae by thermal denaturation and CsCl buoyant density gradient centrifugation reveal a high degree of structural complexity and interspecific heterogeneity. The hepatic taxa exhibit two or more DNA components of varying base composition. Average G4-C contents of total cellular DNA calculated from melting profiles are similarly variable, ranging from 38% to 53% G + C. The green alga Chara , a member of the ancestral line to land plants, shows similarities with liverworts in possessing multiple DNA components of comparable complexity, whereas Hydrodiciyon DNA displays a single component. Detailed hybridization analyses of individual density gradient fractions using α-tubulin, rRNA and ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene probes were performed to locate the low-copy number and moderately repetitive nuclear genes, and the chloroplast chromosome, respectively. The location of each gene within the density gradient is highly variable among the organisms examined; a-tubulin occurs in fractions ranging from 44–64% G + C, rDNA in 50–64% G + C fractions, and the RbcL gene is located in fractions from 30–59% G + C. For a given species, the two nuclear genes normally overlap in their distributions within the gradient. In most instances, neither gene occurs in the major DNA components, indicating that these components may contain repetitive DNAs. The observed variation in the density of the rbcL gene implies substantial reorganization of the chloroplast genome. The overall differences in the genomic components within and between taxa provide insight into the dynamics of DNA structure that have occurred during the extended evolutionary history of these organisms.     

The GC-rich mitochondrial and plastid genomes of the green alga Coccomyxa give insight into the evolution of organelle DNA nucleotide landscape

Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.     

Phylogenetic relationships of Mexican minnows of the genus Notropis (Actinopterygii, Cyprinidae)

We conducted phylogenetic analyses based on complete mitochondrial cytochrome b gene sequences among southern and central Mexican cyprinid species, included in the genera Notropis and Hybopsis. In addition 15 northern species of the genera Notropis and Hybopsis were included in the analyses in order to place the Mexican species into a larger phylogenetic framework. The phylogenetic relationships supported the existence of five major clades: (1) including species of the subgenus Alburnops of the genus Notropis plus N. shumardi; (2) species of the subgenus Notropis; (3) species of the genus Hybopsis; (4) species of the N. texanus + N. volucellus species group of the genus Notropis; (5) Mexican endemic species of the genus Notropis plus the genus Yuriria. Previous phylogenetic inferences based on morphological characters resolved the Mexican minnows analysed as N. sallaei, N. calientis, N. boucardi and Y. alta, non‐monophyletic. According to our cytochrome b evidence all Mexican minnows of the genera Notropis and Yuriria formed a monophyletic group with respect to the northern species of the genera Notropis and Hybopsis. Within the Mexican clade, three well‐supported clades were identified: the first included the closely related species N. moralesi and N. boucardi, which occur in three independent drainages of south Mexico; the second consisted of two different lineages, N. imeldae and an undescribed species of Notropis, inhabiting two independent drainages of south Mexico; the third comprised two central Mexican Notropis species (N. calientis and N. sallaei) and the Y. alta populations. Based on this study and pending a more extensive taxonomic revision of the genus Notropis, we adopt the conservative criterion of considering all Notropis species from southern and central Mexico examined, including Y. alta, as belonging to the genus Notropis. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80 , 323–337.     

Bias in Template-to-Product Ratios in Multitemplate PCR

Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.     

CHLOROPLAST DNA VARIABILITY AMONG LINUM SPECIES

We have compared the chloroplast DNA maps of a selection of Linum species, using recombinant DNA probes derived from L. usitatissimum chloroplast DNA. The heterologous probes have allowed us to construct physical maps for seven other Linum species. The chloroplast DNAs from two other species, L. flavum and L. tenuifolium, were so divergent that restriction maps could not be derived by this method. Analysis of the differences between the chloroplast DNAs has produced a phylogeny separating the species into two groups. These groups are coincident with previous taxonomic groupings and consist of L. perenne-related and non-related species. The major difference between the chloroplast DNAs of the two groups is a 13-kilobase pair segment near an inverted repeat/large single copy region boundary, which is present in the perenne-group species and absent from the non-perenne group species. In addition, we have identified a mutational hotspot analogous to that found in Nicotiana species chloroplast DNAs (Tassopulu and Kung, 1984). Among the species examined, the amount of base pair substitutions approaches 14% for the sites examined.     

Isolation and characterization of major histocompatibility class IIβ genes in an endangered North American cyprinid fish, the Rio Grande silvery minnow (Hybognathus amarus)

The major histocompatibility complex (MHC) is a critical component of the adaptive immune response in vertebrates. Due to the role that MHC plays in immunity, absence of variation within these genes may cause species to be vulnerable to emerging diseases. The freshwater fish family Cyprinidae comprises the most diverse and species-rich group of freshwater fish in the world, but some are imperiled. Despite considerable species richness and the long evolutionary history of the family, there are very few reports of MHC sequences (apart from a few model species), and no sequences are reported from endemic North American cyprinids (subfamily Leuciscinae). Here we isolate and characterize the MH Class II beta genes from complementary DNA and genomic DNA of the non-model, endangered Rio Grande silvery minnow (Hybognathus amarus), a North American cyprinid. Phylogenetic reconstruction revealed two groups of divergent MH alleles that are paralogous to previously described loci found in deeply divergent cyprinid taxa including common carp, zebrafish, African large barb and bream. Both groups of alleles were under the influence of diversifying selection yet not all individuals had alleles belonging to both allelic groups. We concluded that the general organization and pattern of variation of MH class II genes in Rio Grande silvery minnow is similar to that identified in other cyprinid fishes studied to date, despite distant evolutionary relationships and evidence of a severe genetic bottleneck.     

Characterization of heterochromatin in different species of the Scilla siberica group (Liliaceae) by in situ hybridization of satellite DNAs and fluorochrome banding

Satellite DNAs have been isolated from the monocotyledonous plants Scilla siberica, S. amoena, S. ingridae (all are highly GC-rich), and S. mischtschenkoana by using the Ag⁺ –Cs₂SO₄ density centrifugation technique. Hybridization in situ has been performed with ₃H-cRNA to these satellite DNAs in all four species. In each species, the endogenous satellite DNA is located mainly in intercalary and major heterochromatin bands associated with terminal regions and nucleolar organizer regions (NORs) but not in centromeric regions. Patterns observed after cross-species hybridization show a high degree of satellite DNA homology between S. siberica, S. amoena, and S. ingridae. By contrast, satellite DNA of S. mischtschenkoana consists largely of different, non homologous DNA sequences, with two exceptions: (i) the NORs of all four species contain similar satellite sequences, and (ii) a strong homology exists between the satellite DNA of S. mischtschenkoana and centromeric DNA of S. siberica but not with those of S. amoena and S. ingridae. — Heterochromatin has also been characterized by the AT-specific fluorochromes quinacrine (Q) and DAPI and the GC-specific agent chromomycin A₃ (CMA₃), in combination with two counterstaining techniques. While CMA₃-fluorescence is largely in agreement with data on base composition and location of the specific satellite DNAs, the results with Q and DAPI are conflicting. Prolonged fixation has been found to change the fluorescence character in certain instances, indicating that other factors than the base sequence of the DNA also play a role in fluorochrome staining of chromosomes. The results are discussed in relation to the taxonomy and phylogeny of the four species.