Characterization and localization of cryptic satellite DNAs in barley (Hordeum vulgare)

Summary Three satellites on the heavy side of the main band and two satellites on the light side were isolated in a pure from by preparative ultracentrifugation of H. vulgare DNA in Ag⁺/Cs₂SO₄ density gradients. The satellites were characterised in terms of their buoyant densities in CsCl and their thermal dissocation temperature in both native and reassociated forms to Cot 4. In CsCl gradients, heavy satellites formed a single peak whereas light satellites resolved into more than one component. Thermal transitions of some satellites indicated the presence of more than one molecular species. The multicomponent nature of thermal denaturation profiles was evident on differential analysis. Radioactive RNAs complementary to the three heavy satellites of H. vulgare were localised by in situ hybridization onto its nuclei and chromosomes. One heavy satellite (H₃) was found to be distributed on all chromosomes, although one pair showed less hybridization compared to the others. The other satellite (H₁) appeared to be present in a much lower amount on the chromosomes.     

Fractionation and characterization of satellite DNAs of the kangaroo rat (Dipodomys ordii)

Nuclear DNA from liver cells of the kangaroo rat species Dipodomysordii was fractionated and characterized with the aid of buoyant density gradients in neutral and alkaline CsCl and in Ag⁺-Cs₂SO₄. More than one-half of the DNA was present in three density satellites, a greater proportion than in any other species yet reported; the purified satellite DNAs were denser than principal DNA. All satellite fractions revealed sharp isopycnic bands and narrow denaturation profiles. Two had identical buoyant densities but differed substantially in T_m, base composition, and reassociation kinetics. In alkaline CsCl all three satellites, as well as a shoulder of intermediate repetitive DNA on the heavy side of the principal band, revealed unique strand densities. The most highly repetitive satellite was unusually rich in (G + C) and contained 6.7% of 5-methylcytosine. A survey of internal organs and spermatozoa of an adult male revealed no significant differences in distribution of the satellites among tissues.     

Isolation of twelve satellite DNAs from Drosophila hydei

The genome of a Drosophila hydei genotype with a reduced amount of heterochromatin was fractionated by three cycles of preparative gradients: firstly in Ag⁺/Cs₂SO₄, secondly in actimomycin D/CsCl, and finally in neutral CsCl. Using this method, twelve highly repetitive simple-sequence satellites were isolated. Ten of them comprised only a minor amount of the genome in contrast to the two major satellites found earlier¹ (p = 1.696 and 1,714 g/cm³). These minor satellites were characterized by their banding in the gradient systems used, by their density in neutral CsCl, and by their melting point. Using these characteristics, it was found that the fractions of the Ag^?/Cs₂SO₄ gradient do not contain purified single components, because up to five different satellites band in the same position of the Ag^?/Cs₂SO₄ gradient. It was possible to isolate a high number of satellites even from a genome with a reduced amount of heterochromatin. Thus, the D hydei heterochromatin does not domain one unique highly repetitive sequence DNA, but is comprised of many different satellite sequences.     

Two AT-rich satellite DNAs in the chironomid Glyptotendipes barbipes (Staeger)

Two AT-rich satellite DNAs are present in the genome of Glyptotendipes barbipes. The two satellites have densities of 1.680 g/cm³ (=21% GC) and of 1.673 g/cm³ (=13% GC) in neutral CsCl-density gradients. The main band DNA has a density of 1.691 g/cm³ (=32% GC). This value is in agreement with the 33% GC-content of G. barbipes DNA calculated from thermal denaturation (T_M=83° C). — In brain DNA as well as in salivary gland DNA the two satellite sequences together comprise 12–15% of the total G. barbipes DNA. Comparisons of the density profiles of DNA extracted from polytene and non-polytene larval tissue gave no hints for underreplication of the satellite DNAs during polytenization. — The two satellite DNAs have been isolated from total DNA by Hoechst 33258-CsCl density centrifugation and then localized in the polytene salivary gland chromosomes by in situ hybridization. Both satellite sequences hybridize to all heterochromatic centromere bands of all four chromosomes of G. barbipes. Satellite I (1.673 g/cm³) hybridizes mainly with the middle of the heterochromatin, satellite II (1.680 g/cm³) hybridizes with two bands at the margin of the heterochromatin. In situ hybridization with polytene chromosomes of Chironomus thummi revealed the presence of G. barbipes satellite sequences also in the Ch. thummi genome at various locations, mainly the centromere regions.     

Chromosomal localizations by in situ hybridization of the repetitious human DNA families and evidence of their satellite DNA equivalents

Four of the five major repetitious human DNA families, have been mapped by the in situ hybridization technique at their T_OPT values. Two of the lighter density DNA families have autoradiographic grain patterns over heterochromatic chromosomal regions that resemble those of known satellite DNAs. The two heaviest density DNA families have autoradiographic grain patterns of middle repetitious DNAs, with all chromosomes showing labelling. Some evidence suggests that one of these DNA families is concentrated in certain chromosomal regions. Both DNA families exhibit biphasic T_OPT curves. The presence of two thermal stability classes of hybrids suggests sequence interspersion. By co-enrichment studies in Ag⁺-Cs₂SO₄ gradients, evidence suggests the origin of the three lightest density renaturated human DNA families to be satellites I, II and III.     

Isolation and characterization of satellite DNA from mustard seedlings

The DNA from mustard (Sinapis alba L.) seedlings was examined by neutral CsCl and Ag⁺/Cs₂SO₄ density gradient centrifugation. Different satellite fractions were revealed by these two methods. The satellite fractions obtained from the Ag⁺/Cs₂SO₄ density gradient could not be generally correlated with satellite DNA fractions observed in CsCl. In CsCl density gradient centrifugation, a main band at density 1,695 g/cm³ and a heavy shoulder at density 1,703 g/cm³ are found. By preparative CsCl gradient centrifugation the heavy shoulder can be enriched but not completely separated from the main band DNA.—Gradient centrifugation by complexing the DNA with Ag⁺ rf. 0.25 to DNA phosphate reveals three distinct fractions which are further characterized: The heavy satelite DNA fraction revealed by Ag⁺/Cs₂SO₄ gradient centrifugation has the same density in a CsCl gradient and the same Tm value as the main band, but differs from main band DNA in the details of its melting profile and in its renaturation kinetics. The light Ag⁺/Cs₂SO₄ satellite DNA fraction had a higher melting temperature corresponding to a GC-rich base composition. Differences between these 3 fractions are observed in thermal denaturation and renaturation profiles, hybridization in situ with ribosomal RNA, and their response to restriction endonuclease digestion. The light satellite fraction from the Ag⁺/Cs₂SO₄ gradient, rich in ribosomal cistrons corresponds to the heavy shoulder DNA of neutral CsCl gradients which also is rich in ribosomal cistrons. The heavy satellite fraction from Ag⁺/Cs₂SO₄ gradient which contains highly repetitive short nucleotide sequences could not be revealed by the classical CsCl gradient centrifugation technique.     

Analysis and in situ hybridization of cryptic satellites in Hordeum arizonicum

Summary Three satellites, one (H₁) on the heavy side of the main band of Hordeum arizonicum DNA and two (L₁, L₂) on the lighter side were purified using preparative silver-cesium sulphate density gradients. The native and the reassociated satellite DNAs were analysed in terms of buoyant densities and thermal dissociation. In cesium chloride gradients the H₁ and L₁ satellites formed single peaks corresponding to buoyant densities of 1.700 and 1.701 g · cm^–3 respectively while the L₂ satellite gave two peaks (1.680 and 1.661 g · cm^–3). The H₁ satellite showed three thermal components (Tm=82.5 °C, 87 °C and 91.5 °C) while the L₁ and L₂ had three (86.5, 92, 97.5 °C) and two (86, 95 °C) respectively. The H₁ satellite was localized on the nuclei and chromosomes. The distribution of H₁ onto approximately on third of the complement may reflect the genome specific origin of this satellite.     

The effect of natural selection on enzymic catalysis.

The karyotype of Drosophila nasutoides reveals a very large autosome pair at the metaphase plate. The application of the C-banding technique shows that this chromosome is almost entirely heterochromatic and an isochromosome (Cordeiro et al., 1975). Examination of the DNA isolated from purified nuclei of D. nasutoides in neutral CsCl gradients reveals four major satellites. As much as 60% of the total DNA appears as satellites in the DNA from larval brains. The buoyant densities of the four satellites, designated as I through IV in the order of descending density, are 1.687, 1.682, 1.669 and 1.665 g/cm³, respectively. All four satellites show strand separations in alkaline CsCl gradients with the least separation in satellite III. Thermal denaturation studies with purified native satellites show that satellites I and IV consist of repeats of identical sequences, whereas satellites II and III show a large sequence variation between repeating units. As much as 10 to 24% base-pair mis-matching is observed in the reassociated satellite II. The sequence complexities obtained from DNA reassociation kinetics data are 5, 103, 2.3 × 10⁶ and 46 nucleotide pairs for the satellites I, II, III and IV, respectively. The complexity of satellite III is almost as large as that of Escherichia coli, when the reassociation rate is corrected according to the amount of mis-matching in this satellite. All four satellite sequences are localized in one chromosome (dot chromosome) according to in situ hybridizations to polytene chromosomes. The large heterochromatic chromosome seen at the metaphase plate appears as the dot chromosome after polytenization. Therefore, the large heterochromatic chromosome contains all four satellite DNA components.     

Synthesis of structurally unstable type III procollagen in patients with cerebral artery aneurysm

The biosynthesis of collagen was studied in skin fibroblast cultures established from 11 patients with cerebral artery aneurysms. Six patients had familial subarachnoid hemorrhage (SAH), while five patients were considered as sporadic cases. The structural stability of the triplet-helical medium procollagen was studied by measuring the thermal denaturation temperature (T_m) of type I and type II procollagen molecules. Structural instability of type III procollagen was demonstrated in two patients with familial SAH. Te T_m of type III procollagen was 39.0°C and 39.5°C in two of the cell lines, while the control value was 40.3°C. The stability of type I procollagen did not differ from that of the controls, and the main features of the biosynthesis of collagen were similar in the aneurysm patient cell lines and in the controls. The results suggest that a structural defect of type III procollagen may serve as an etiological factor in the formation of cerebral artery aneurysms.     

A study of sequence homologies in four satellite DNAs of man.

Satellites I, II, III and IV (Corneo et al., 1968,1970,1971) have been purified from human male placental DNA. The sequences present in these four DNA components have been characterized by analytical buoyant density, thermal denaturation, DNA reassociation, DNA hybridization and gel electrophoresis coupled with hybridization following either HaeIII or EcoRI restriction endonuclease digestion. Satellites III and IV were found to be virtually indistinguishable by a variety of criteria. Cross-satellite reassociation showed that 40% of the molecules present in satellite III contain sequences that are homologous to 10% of the molecules of either satellite I or satellite II. Reassociated satellite I melts as a single component, as do the hybrid duplexes between satellite I and satellite III. In contrast, reassociated satellites II, III and IV, and the hybrid duplexes formed between satellites II and III and between satellites II and IV, melt as two distinct components with different thermal stabilities.Digestion of satellite III with HaeIII gives rise to a series of fragments whose sizes are 2, 3, 4, 5, 6, 7, 8 and 11 times the size of the smallest 0.17 × 10³ basepair fragment, in addition to a 3.4 × 10³ base-pair male-specific fragment (Cooke, 1976) and high molecular weight material. The sequences contained in the fragments of the HaeIII ladder are diverged from each other as well as being non-homologous with those of the 3.4 × 10³ base-pair and high molecular weight fragments. The latter contain EcoRI recognition sites. Satellite II has a similar pattern of fragments to satellite III following digestion with HaeIII, although it can be distinguished from satellite III on the basis of the products of EcoRI digestion. Satellite I contains neither HaeIII nor EcoRI recognition sites. The cross-satellite homologies of the sequences present in fragments of differing sizes produced by restriction enzyme digestion have also been studied.     

Nucleotide clusters in deoxyribonucleic acids. XII. The distribution of 5-methylcytosine in pyrimidine oligonucleotides of mouse L-cell satellite DNA and main band DNA.

Mouse L-cell DNA radioactively labeled in the 5-methylcytosine (5-MeC) residue was fractionated into satellite and main band DNA. Satellite DNA was found to contain about four times the molar concentration of 5-MeC than the main band DNA. Based on the known 5-MeC content of total L-cell DNA it was calculated that satellite DNA contains 3.5 – 4.6% 5-MeC. Both DNA fractions were depurinated and the pyrimidine oligonucleotides released separated by ionophoresis-homochromatography. In satellite DNA 5-MeC is distributed non-randomly. About 40% of the total 5-MeC is present in the sequence Pu - 5-MeC - Pu. The remainder occurs in the oligonucleotides CT, CT₃, C₂T₄, C₂T₅ and C₃T₅ only. The distribution of 5-MeC in main band DNA differs from that in satellite DNA indicating that two different fractions of the same nuclear DNA are methylated in different sequences.     

The chromosomal distribution of satellite DNA in the germ-line and somatic tissues of the gall midge,Heteropeza pygmaea

Somatic DNA from Heteropeza pygmaea separated in CsCl gradients into a main band DNA (?=1.685 g/cm³) and a satellite band (?=1.716 g/cm³) comprising 15% of the total DNA. The satellite melted sharply at 93.0°C in SSC, 10.4°C higher than the main band DNA. Satellite DNA reassociated rapidly, banding in CsCl heavier than native satellite but lighter than denatured satellite. The complementary strands of the satellite formed a single band in alkaline gradients and hence are apparently similar in G+T composition. — Filter hybridization experiments with Xenopus ribosomal RNA showed that the satellite band does not contain ribosomal cistrons. — Complementary RNA (cRNA) transcribed in vitro from isolated satellite bound extensively to satellite DNA but not to main band DNA. — The strain of Heteropeza used here contained about 58 chromosomes in germ-line cells and reproduced only paedogenetically. During early cleavage, the presumptive somatic nuclei eliminate most of their chromosomes (E-chromosomes) and retain only ten (S-chromosomes). In situ hybridizations with satellite-cRNA showed satellite DNA (prepared from predominately somatic tissues) to be localized in the centromeric heterochromatin of S- and E-chromosomes. Silver grain comparisons suggested that the amount of satellite is equivalent in both types of chromosomes. — Lastly we found that both diploid and polyploid cells contain similar amounts of satellite DNA. We interpreted this to mean that during polyploidization, as has been demonstrated during polytenization, satellite DNA does not replicate or replicates only slightly while other DNA fractions increase.     

Polyacrylamide gel as a medium for DNA dissociation and reassociation

Several properties of thermal denaturation and renaturation of DNA in polyacrylamide gels were investigated: (1) Following electrophoresis the DNA band was scanned and shown to increase in absorbance with increasing temperature. The increase was proportioned to DNA concentration across the peak. (2) The dependence of theT _m on salt concentration over a hundred fold range was similar to that found for DNA in free solution. (3) Denaturation of several DNA samples ranging in G+C content from 26 to 71% was compared in gels and free solution. The relationship betweenT _m and % G+C was virtually identical for both sets of DNAs. (4) The kinetics of DNA renaturation in the gel was followed. Reassociation of bacteriophageT ₄ DNA was 2nd order and proceeded more rapidly in polyacrylamide gels than in free solution.     

Microcalorimetric Investigation of DNA,poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] Melting in the Presence of Water Soluble (Meso tetra (4 N oxyethylpyridyl) Porphyrin) and its Zn Complex

Abstract

Thermodynamic parameters of melting process (δH_m, T_m, δT_m) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C))·poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased T_m of satellite fraction by 24°C, but ZnTOEpyp(4), on the contrary, predominately bound with AT-rich sites and increased DNA main stage T_m by 18°C, and T_m of poly(dA)poly(dT) increased by 40 °C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes—strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode—strong binding—took place. The weak binding is characterized with shifting of T_m by some grades, and for the strong binding T_m shifts by ～ 30–40°C. Invariability of ΔH_m of DNA and poly(dA)poly(dT), and sharp increase of T_m in the range of r from 0.08 to 0.25 for thymus DNA and 0.01–0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H₂O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width ΔT_m caused by increase of added ZnTO- Epyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which ΔT～T_GC-T_AT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193–205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.     

Isolation and characterization of DNA from the entomopathogenBeauveria bassiana

Beauveria bassiana chromosomal (chr) and mitochondrial (mt) DNA were isolated, purified, and characterized. Modification of a recent rapid method of total DNA extraction was followed by DNA purification on CsCl-bisbenzimide gradients. Cytosine methylation at 5′-CpG-3′ doublets was undetectable in the total DNA as demonstrated byMspI andHpaII restriction enzyme digests. The chrDNA had a bouyant density of 1.7130 g/ml (G + C ratio of 53.4%) and a T_m of 92.6°C (G + C ratio of 56.9%). Mitochondrial DNA was isolated from total DNA or purified mitochondria and had a buoyant density of 1.7120 g/ml (G + C ratio of 52.3%) and a biphasic DNA melting curve. A minor melt at 76.2°C (16.8% G + C ratio) yielded a 10% increase in absorbance while the major melt at 93.1°C (58% G + C ratio) yielded a 30% increase in absorbance. The mtDNA was estimated by gel electrophoresis to be 17.6 ± 0.6 MDa and determined to be 28.5 kb by restriction enzyme analysis. Electron microscopy of the mtDNA revealed circular molecules having an average contour length of 8.84 ± 0.51 μm. A physical map of the mtDNA was generated by double restriction enzyme digestion using the restriction enzymesClaI,EcoRI,SalI,BstEII, andXhoI.     

Sequence of the cryptic satellite DNA from the plant Sinapis alba

A satellite DNA with a buoyant density equal to that of main band DNA in neutral cesium chloride (‘cryptic satellite’) can be isolated from the DNA of mustard (Sinapis alba) nuclei by Ag⁺/Cs₂SO₄ density gradient centrifugation. This satellite is cleaved into 172 bp repeat units by HinfI, AluI or HaeIII. The HinfI fragments have been further cleaved by AluI, and seven AluI subfragments have been sequenced. As a result two versions of a basic 172 HinfI repeat have been found, one (A + B) with an additional HinfI site. These two sequences (A + B and C) are the most frequent versions of the basic repeat of mustard satellite DNA. The basic 172 bp unit does not contain subrepeats or palindromic sequences. It is not similar (at a criterion of 15 common bases) with any known satellite sequence. It is not unusually highly methylated in the native state.     

Chloroplast ribosomal RNA genes in the chloroplast DNA of Euglena gracilis

Euglena chloroplast DNA has a buoyant density in CsCI of 1.686. Shearing this DNA produces a satellite band at density 1.700. The satellite, easily lost during preparative CsCI gradient centrifugation of chloroplast DNA, contains the genes for chloroplast ribosomal RNA. Pure Euglena chloroplast DNA is shown to contain one set of ribosomal RNA genes for each 90 × 10⁶ daltons of DNA.     

Variation in satellite DNA from some higher plants

Pure satellite DNAs were prepared as minor components after centrifugation to equilibrium on CsCl gradients. A single satellite DNA band was isolated from flax (Linum usitatissimum) DNA and two bands were resolved in cucumber (Cucumis sativus) DNA. These apparently homogeneous components of the plant genomes were further analyzed by thermal denaturation and renaturation. The flax satellite DNA appeared homogeneous on thermal denaturation but was shown to contain several components of renaturation. The two cucumber satellite DNAs were different from each other, but both showed at least two components in denaturation and renaturation analyses. Renaturation in the three satellites, particularly in flax, was inaccurate, indicating a considerable degree of sequence divergence. Although each satellite contained quite large amounts of simple repetitious sequences, a residual heterogeneous DNA fraction was always present. It is considered that this was too large a portion of the satellite DNA to be due to organelle or ribosomal DNA in cucumber. The latter possibility is precluded in flax, where the satellite is completely resolved in buoyant density from both organelle and ribosomal DNA.     

Analysis of DNA from related and diverse species of Barley

DNAs of three closely related diploid species Hordeum vulgare, H. agriocrithon and H. spontaneum were compared among themselves and with a group of three species related to each other H. californicum (2x), H. jubatum (4x), and H. arizonicum (6x) having one genome in common.Buoyant densities of the DNAs from these species fall within a narrow range (1.700–1.701 g cm⁻³). Melting temperatures (T_m) of the DNAs are also similar (85.2–86.2°C). None of the DNAs showed any satellite band in neutral Cesium chloride gradients. In silver-cesium sulphate gradients, however, DNAs of all the species formed satellites on both the light and heavy sides of the main. band The diploid series have similar but not identical satellite patterns. The members of the polyploid series showed similar satellite patterns among themselves but distinct from those of the diploid series. In the polyploid group the amount of satellite showed a progressive decrease with the increase in the ploidy.The amount of repeated DNA (reassociating up to Cot 100) in H. vulgare was 69% and in H. arizonicum 65% of the total. The two species differed in their Cot curves. The low Cot fractions of H. arizonicum contained components of low buoyant densities which were absent in the corresponding Cot fractions of H. vulgare DNA.