Non-repetitive DNA sequence divergence in phylogenetically diploid and tetraploid teleostean species of the family cyprinidae and the order isospondyli

Non-repetitive DNA of anciently tetraploid teleostean species was analysed for the presence of duplicated sequences. Closely related diploid species were investigated in comparison. From the reassociation kinetics of total nuclear DNA, rate constants and fraction sizes of classes of repetitive and non-repetitive sequences were determined. DNA fractions enriched in the slowest renaturing sequence class were prepared and subjected to reassociation. The rate constants of these reactions were compared with the values expected for single-copy DNA from analytical genome size determinations. From reassociated DNA enriched in non-repetitive sequences also the melting temperatures were determined as a measure of internal base sequence heterogeneity. It has been shown that the two ancient tetraploids Cyprinus carpio and Thymallus thymallus are, with regard to the thermal stability of reassociated non-repetitive DNA, and with regard to the correspondence of reaction rates with the values expected for single copy DNA, indistinguishable from diploid controls (Rutilus rutilus, Clupea harengus and Sprattus sprattus). The tetraploid species Salmo irideus, Salvelinus fontinalis and Coregonus lavaretus appear as very recent tetraploids with regard to these criteria. The significance of the results for estimating the time of occurence of polyploidisation events in these taxa is discussed.     

Rodent DNA reassociation kinetics

The kinetics of reassociation of the nuclear DNA of eight rodents has been examined to determine the amount and repetition frequency of various DNA fractions. Each rodent had at least two distinct, repeated DNA fractions, one group repeated about 300 times and the other repeated about 50,000 times. As shown, the actual repetition frequencies and the amount of the various DNA fractions varied from one species to another. Genetic implications of these results are discussed.     

Fluorometric measurement of DNA reassociation kinetics.

A new radioimmunoassay (RIA) method is proposed, utilizing cellulose disks coated with a ${}^{125}\text{I-}\text{Ag}\text{Ab}$ complex as a single reagent. The labeled antigen is released into the incubation medium proportionally to the concentration of the corresponding antigen in the sample. The method is comparable to the classical RIA with regard to sensitivity, precision, and specificity, but it has the advantage of a wide range of nondependence on the sample volume and greater practicability (the analysis is performed using a single-step procedure). The assay of human chorionic somatomammotropin is adopted as a model.     

DNA reassociation kinetics and hybridization in different angiosperms

Reassociation kinetics ofDaucus carota andPetroselinum crispum (Apiaceae), andDatura innoxia (Solanaceae) are presented. Hybridization of³H-labelled DNA of two carrot cultivars indicate strong qualitative homologies of DNA sequences; nevertheless, certain quantitative differences in some Cotregions seem to exist. However, homologous sequences ofDaucus DNA with DNA ofDatura, and, suprisingly, even with DNA ofPetroselinum are very restricted: between 8% in the repeated regions and ca. 7–9% in the unique regions.     

Length dependence in reassociation kinetics of radioactive tracer DNA.

The reassociation kinetics have been measured for radioactive Escherichia coli DNAs (tracers) of various average single-strand lengths reassociated alone and in the presence of excess unlabeled DNA (driver) of two different average lengths. Hydroxylapatite binding was used to follow the reaction time course. The length-dependence of the rate constant determined in the tracer self-reassociation reactions is in agreement with the square-root dependence previously determined (Wetmur, J. G., & Davisond, N. (1968) J. Mol. Biol. 31, 349-370) using optical methods to follow the time course. However, for the driver-tracer reactions, where the radioactive DNA reassociates largely with DNA of a different average length, the dependence of the rate constant upon average tracer length is increased and approaches an L to the first power dependence. In 0.18 M Na+, the variation of the rate constant for tracer reassociation with the lengths of the reassociating strands has been shown to fit the simple equation k = (9.0077).(L T 0.55 + 1/L D 0.55), where k is the observed rate constant in L mol-1 s-1 and L(T)and L(D) are the weight average tracer and driver lengths, respectively, in nucleotides. This dependence suggests that the rate of nucleation of two free strands is proportional to the sum of the reciprocals of the hydrodynamic radii of the two strands.     

DNA reassociation kinetics of closely related species

The kinetics of reassociation of the DNA of three groups of closely related organisms were examined. The laboratory mouse was compared to an Asiatic mouse, whose chromosome number is the same but whose chromosome organization is different. Chinese hamster (2N=22) was compared to Syrian hamster (2N=44), and Haplopappus gracilis (2N=4) was compared to H. ravenit (2N=8). It was found that the most highly repeated DNA fractions of the three comparative sets of organisms differ in their reaction rates. However, these fractions of the related hamsters, haplopappi, and probably the mice, do not differ in the amount of DNA composing the fractions. The intermediately fast reassociating DNA and the unique DNA do not differ between members of related pairs of organisms. The implication of these results is that a short sequence of DNA may be highly copied in one organism, while in a related organism a longer DNA sequence is repeated a fewer number of times, and the total amount of repeated DNA may be the same in both related organisms.     

Cytoplasmic DNA: Differentiation by reassociation kinetics

Rat liver nuclear and cytoplasmic DNA samples were denatured and the kinetics of their reassociation was measured. About 85% of the soluble cytoplasmic (mitochondrial) DNA reannealed rapidly with a $\text{C}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.03}$ while 65% of the particulate (microsomal) DNA reassociated with a $\text{C}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.14}$ Both nucleic acids were clearly differentiated from nuclear DNA in their reassociation kinetics. The results indicate that both mitochondrial and microsomal DNA consist mainly of single components or closely related families with repetitive sequences.     

Correlation between animal radiosensitivity and kinetics of DNA reassociation

A study was made of DNA of different animal species with various radiosensitivity: guinea pig, man, rat and rabbit. It was shown that the radioresistance of the organism increases with increasing relative content of a rapidly reassociating fraction in DNA. As the kinetic complexity of moderately repetitive sequences increases and the total percentage of meaddle and unique DNA fractions grows, the radioresistance of the organism decreases.     

Single-copy DNA relationships between diploid and tetraploid teleostean fish species

The degree of single-copy DNA relatedness among nine Salmonid, Osmerid, and Clupeid species (teleosts, order Isospondyli) was explored by interspecific DNA hybridization and the determination of the thermal stability of these hybrids. It is shown that the extent of base substitution and the amount of shared sequences is largely consistent with the systematic interrelationship of the species compared. A tentative estimate of the average base substitution rate is about 0.1–0.25% per million years, which is in the range typical for animal and plant nuclear genomes. The results are also discussed in view of the phylogenetically tetraploid state of the Salmonid genomes. A comparison of the amount of intra-genomic and inter-genomic divergence in the tetraploids suggests that a polyploidization event occurred recently in Salmonid evolution.     

Preparation of radioiodinated simian virus 40 DNA for use in DNA - DNA reassociation kinetics experiments.

A method is described for the preparation of 125I-labelled SV40 DNA. Using this method, SV40 DNA can be routinely labelled to 15 - 10(6) dpm per mug; much higher specific activities are easily obtained by minor modifications of the method. Once incorporated, the radioactive label dissociates from DNA exceedingly slowly at 4 degrees C or at 68 degrees C. Iodinated SV40 DNA is shown to be useful in the quantitation of viral nucleic acid sequences in SV40-transformed 3T3 cells by DNA - DNA reassociation kinetics.     

A rapid computer analysis for multicomponent DNA reassociation kinetics

An iterative program, Gaushaus, has been adapted to analyze complex DNA reassociation kinetics. With valid experimental data, rapid convergence to a solution is always achieved by this program even when the starting parameters are grossly in error. The output of the program also includes useful statistical information.     

RFLP variation in diploid and tetraploid alfalfa

Summary Alfalfa (Medicago sativa L.) is a major forage crop throughout the world. Although alfalfa has many desirable traits, continued breeding is required to incorporate pest resistances and other traits. We conducted this study to determine the amount of restriction fragment length polymorphism (RFLP) variability present within and between diploid and tetraploid alfalfa populations, and whether or not this variability is sufficient for construction of an RFLP map. Diploid plants from M. sativa ssp. falcata, ssp. coerulea, and ssp. sativa and tetraploid spp. sativa cultivars Apollo, Florida 77, and Spredor 2 were included. A total of 19 cDNA clones was probed onto genomic Southern blots containing DNA digested by EcoRI, HindIII, or BamHI. Phylogenetic trees were produced, based on parsimony analysis of shared restriction fragments. Evidence for extensive gene duplication was found; most probes detected complex patterns of restriction fragments. Large amounts of variation are present within all diploid subspecies. M. sativa ssp. falcata plants formed clusters distinct from ssp. sativa or ssp. coerulea plants, which were not distinctly clustered. Some M. sativa ssp. falcata plants were more similar to the other groups than to other plants within ssp. falcata. Variation among tetraploid cultivars showed that Florida 77 and Apollo had more similarities than either showed with Spredor 2. All three cultivars showed large within-population variation, with Apollo being the most diverse and Spredor 2 the least. Based on these results, development of an RFLP map at the diploid level appears possible. Also, differentiation of cultivars, particularly ones of divergent origin, seems possible based on RFLP patterns.     

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).     

Application of DNA sonication in the study of reassociation kinetics]

Single-stranded breaks arising is DNA under sonication divide the molecule in short double-stranded regions of lengths depending on sonication conditions. The reassociation rate of DNA with single-stranded breaks is considerably lowered as compared with intact DNA of the same length. For sonicated DNA the slowing of reassociation and the deflection to higher orders of C(o)t of the reaction is observed at the late stages. Further process of sonication occurs likely on single-stranded breaks and DNA fragments obtained as a result of infinite sonication are practically intact. Reassociation of DNA, degraded to a "saturation", proceeds as a second order reaction in precise accordance with the obtained fragment length.     

DNA reassociation kinetics in relation to genome size in four amphibian species

DNA reassociation kinetics were studied, by means of the hydroxyapatite chromatography method, for four species of Amphibians with different nuclear DNA content: Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) of the Anura subclass and Trituras cristatus (23 pg) and Necturus maculosus (52 pg) of the Urodela subclass.Within each subclass the two species studied were found to have about the same absolute amount of unique DNA. The differences of total nuclear DNA can be accounted for by quantitative variations of the repetitive sequence classes, at least in part due to changes in the number of copies of the various sequences. On the contrary the great difference in nuclear DNA between the two subclasses, Anura and Urodela, involves all sequence classes in parallel; the slowly reassociating fraction appears to be unique in spite of a tenfold difference in absolute amount.The dependence of reassociation kinetics on DNA fragment length for the four species indicates for all of them an interspersed organization of the various sequence classes.     

Chromosomal DNA denaturation and reassociation in situ.

The degree of chromosomal DNA (cDNA) denaturation and renaturation on polytene chromosomes has been measured by UV microspectrophotometry. Also DNA losses occurring upon denaturation have been quantified by Feulgen, gallocyanin-chromalum and UV. It has been observed that denaturation in alkali (0.07 N NaOH at room temperature) and formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C removes about 30% of the DNA. Low DNA loss occurs upon denaturation in HCl (0.24 M) at room temperature and 60% formamide: 2 × 10^?4 M EDTA (pH 8) at 55 °C. The presence of 4% formaldehyde in the denaturation buffer prevents DNA loss. After denaturation of chromosomes in 0.1 × SSC containing 4% formaldehyde at 100 °C for 30 sec, an hyperchromicity of 39 °C is observed. The denaturation efficiency varies with the denaturation treatment. The percentage reassociation was measured from the difference in the UV absorption of renatured chromosomes and that of denatured chromosomes from the same set. It seems that in our conditions DNA:DNA reassociation does not occur. The efficiency of hybridization is proportional to the denaturation extent of the DNA. However, the entire fraction of DNA which has been denatured is not available for hybridization.     

Simple sequence repeat diversity in diploid and tetraploid Coffea species.

Thirty-four fluorescently labeled microsatellite markers were used to assess genetic diversity in a set of 30 Coffea accessions from the CENICAFE germplasm bank in Colombia. The plant material included one sample per accession of seven East African accessions representing five diploid species and 23 wild and cultivated tetraploid accessions of Coffea arabica from Africa, Indonesia, and South America. More allelic diversity was detected among the five diploid species than among the 23 tetraploid genotypes. The diploid species averaged 3.6 alleles/locus and had an average polymorphism information content (PIC) value of 0.6, whereas the wild tetraploids averaged 2.5 alleles/locus and had an average PIC value of 0.3 and the cultivated tetraploids (C. arabica cultivars) averaged 1.9 alleles/locus and had an average PIC value of 0.22. Fifty-five percent of the alleles found in the wild tetraploids were not shared with cultivated C. arabica genotypes, supporting the idea that the wild tetraploid ancestors from Ethiopia could be used productively as a source of novel genetic variation to expand the gene pool of elite C. arabica germplasm.