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1.
Location of Satellite and Homogeneous DNA Sequences on Human Chromosomes 总被引:15,自引:0,他引:15
HUMAN DNA1,2 contains at least two satellite fractions—satellite I (0.5% of the genome) which bands at a density of 1.687 g/cm3 in neutral CsCl and satellite II (2% of the genome) which bands at 1.693 g/cm3. The main band DNA has an average buoyant density between 1.670 and 1.720 g/cm3 and a light shoulder (constituting 15% of the genome) with a buoyant density of 1.696 g/cm3, referred to as homogeneous mainband. Satellite DNA has been observed in many higher organisms3, usually with an unknown function, notable exceptions being cistrons coding for ribosomal RNA4 and the DNA coding for histone messenger RNA5. To investigate possible functions of human repetitive DNA we have studied the annealing of complementary RNA fractions to chromosomes using in situ hybridization6,7. We describe here preliminary observations using human satellite II and homogeneous mainband fractions. 相似文献
2.
Embryo DNA, isolated from ungerminated seeds of Lupinus angustifolius L., contains an exceptionally high amount of guanine-cytosine-rich satellite DNA. The thermal denaturation curve of total embryo DNA is biphasic with an inflexion point at 62% denaturation, indicating the presence of satellite DNA. The satellite fraction could be separated from the mainband DNA by three successive preparative CsCl-gradient centrifugations. The densities of the DNA fractions are 1.7045 g cm-3 and 1.6925 g cm-3, respectively. The percentages of guanine-cytosine calculated from these densities are comparable to the percentages of GC calculated from the melting temperatures. Finally, ressociation studies prove that foldback DNA and highly repeated sequences are much more frequent in the satellite DNA fraction than in the mainband DNA.Abbreviation
C
o
t
the product of the DNA concentration (mol nucleotides l-1) and the time (s) of incubation in a DNA reassociation reaction
- GC
guanine-cytosine
- np
nucleotide parirs
- T
temperature interval between 16 and 84% denaturation 相似文献
3.
Characterization of deoxyribonucleic Acid species from castor bean endosperm: inability to detect a unique deoxyribonucleic Acid species associated with glyoxysomes
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Three DNA buoyant density species (nuclear, 1.692 g cm−3; mitochondria 1.705 g cm−3; and proplastid, 1.713 g cm−3) can be detected in extracts from castor bean endosperm. No other buoyant density species can be identified. DNA extracts from sucrose density gradient purified glyoxysomes exhibit varying amounts of each of the three identified DNAs but no other distinguishable DNA species. RNA synthesized in vitro by Escherichia coli RNA polymerase using purified castor bean nuclear DNA as a template, hybridizes equally well with its template and with the 1.692 g cm−3 species from glyoxysome fractions. These results are discussed in terms of their relevance to microbody biogenesis. 相似文献
4.
M. Steinemann 《Chromosoma》1978,66(2):127-139
The DNAs of five Chironomus species, C. plumosus, C. nuditarsis, C. thummi thummi, C. melanotus, and Camptochironomus pallidivittatus, were investigated in analytical neutral isopycnic CsCl density gradients. DNA was isolated both from larval brains (diploid-DNA) and salivary glands (polytene-DNA). The buoyant densities of mainband DNAs were 1.692 g/cm3, with the exception of C. melanotus whose mainband had a density of 1.693 g/cm3. The densities correspond to a calculated GC content of 33% and 34% respectively. Only in C. melanotus was the DNA clearly resolved into mainband DNA and two distinct satellite shoulders: Satellite I, with a buoyant density of 1.684 g/cm3 (25% GC, calculated) and satellite II of 1.679 g/cm3 (19% GC, calculated). The two satellites comprise 15% of the total DNA in the diploid-DNA and they also occur in the polytene-DNA, where they make up 11%. The results are discussed in the general context of under- and overreplication in polyploid and polytene cells. 相似文献
5.
Reciprocal induction of cell division by cells of complementary mating types in Tetrahymena 总被引:1,自引:0,他引:1
J Wolfe 《Experimental cell research》1974,87(1):39-46
Chick embryo fibroblasts in monolayer culture were synchronized by contact inhibition and serum starvation. Nuclear DNA isolated from the [3H] thymidine pulse-labelled cells throughout the period of DNA synthesis (S phase) was analysed by hydroxylapatite chromatography after renaturation at different C0t values. It is shown that repeated sequences having different frequencies of reassociation, replicate differently throughout the S period. In order to study the distribution of the repeated sequences, DNA isolated during the S period was fractionated according to its buoyant density. It is shown that only some of the highly reiterated sequences which are included in the high buoyant density DNA fractions, replicate equally well during the early and the late S periods. By contrast, reiterated sequences of the low buoyant density DNA fractions replicate mainly during the late S period. 相似文献
6.
Condensed and dispersed chromatin fractions were isolated from human placental nuclei. The DNA of each fraction was purified and characterised by isopycnic centrifugation, thermal fractionation on hydroxylapatite (HAP) and sequence complexity studies. The DNAs had identical buoyant densities in neutral CsCl (1.698 g/cm3) and similar melting profiles on HAP. Analytical ultracentrifugation in Ag+-Cs2SO4, however, showed that satellite DNAs were present in the condensed fraction DNA (DNAC) but were not visible in the dispersed fraction DNA (DNAD). In addition, DNAC was found to be enriched in highly reiterated sequences (20% reassociated by C0t 10?3) which can be correlated with the presence of satellite DNAs, whereas DNAD contained only 3% of these fast reassociating sequences. In contrast DNAD contained 30% intermediate sequences (reassociating between C0t 10?3 and C0t 100) which represent only 10% of DNAC. The reassociated highly repeated sequences of DNAC showed the presence of two components in both CsCl density gradients and HAP thermal elution studies. This suggests that either there are sequence relationships resulting in partial mismatching between the different highly repeated DNA sequences in this fraction, or that highly repeated sequences are associated with less repetitious DNA. The results are discussed in terms of possible differences in genetic activity between the chromatin fractions. 相似文献
7.
Calf DNA preparations having molecular weights of 5 to 7 × 106 have been fractionated by preparative Cs2SO4—Ag+ density gradient centrifugation into a number of components. These may be divided into three groups: (1) the main DNA component (1.697 g/cm3; all densities quoted are those determined in CsCl density gradients), the 1.704 and 1.709 g/cm3 components form about 50, 25 and 10% of the genome, respectively; they are characterized by having symmetrical CsCl bands and melting curves, both of which have standard deviations close to those of bacterial DNAs of comparable molecular weight, and by their G + C contents being equal to 39, 48 and 54%, respectively; after heat-denaturation and reannealing, their buoyant densities in CsCl are greater than native DNA by 12, 10 and 3 mg/cm3, respectively. (2) The 1.705, 1.710, 1.714 and 1.723 g/cm3 components represent 4, 1.5, 7 and 1.5% of the DNA, respectively, and exhibit the properties of “satellite” DNAs; their CsCl bands and melting curves have standard deviations lower than those of bacterial DNAs; after heat-denaturation and reannealing, their buoyant densities are identical to native DNA, except for the 1.705 g/cm3 component, which remains heavier by 5 mg/cm3; in alkaline CsCl, only the 1.714 g/cm3 component shows a strand separation. (3) A number of minor components, forming 1% of the DNA, have been recognized, but they have not been investigated in detail; two of them (1.719 and 1.699 g/cm3) might correspond to ribosomal cistrons and mitochondrial DNA, respectively. 相似文献
8.
Satellite DNA associated with heterochromatin in Rhynchosciara 总被引:8,自引:0,他引:8
The DNA of Rhynchosciara hollaenderi was examined using isopycnic centrifugation in neutral CsCl. Two low density minor bands (collectively termed satellite DNA) were detected in addition to the main band DNA. Main band DNA has a buoyant density of 1.695 g/cm3. The larger of the two minor bands has a buoyant density of 1.680 g/cm3 while the smaller of the two minor bands has a buoyant density of about 1.675 g/cm3. Thermal denaturation studies have confirmed the presence of the two minor classes of DNA.—The satellite and main band DNAs were isolated in relatively pure form and were transcribed in vitro using DNA-dependent RNA polymerase from Escherichia coli. Annealing of the two complementary RNAs (cRNAs) with main band and satellite DNA was examined using filter hybridization techniques.—The chromosomal distribution of the satellite DNA was determined by in situ molecular hybridization of satellite-cRNA with Rhynchosciara salivary gland chromosomes. Satellite-cRNA hybridized with the centromeric heterochromatin of each of the four chromosomes (A, B, C, and X) and with certain densely staining bands in the telomere regions of the A and C chromosomes. Main band-cRNA annealed with many loci scattered throughout the chromosomes including areas containing satellite DNA. 相似文献
9.
Erwin R. Schmidt 《Chromosoma》1980,79(3):315-328
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 (TM=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. 相似文献
10.
Mary Lake Polan Susan Friedman Joseph G. Gall Walter Gehring 《The Journal of cell biology》1973,56(2):580-589
Mitochondrial DNA (MtDNA) with a neutral buoyant density of 1.681 g/cm3 has been isolated from unfertilized eggs of Drosophila melanogaster. This DNA is a circular molecule with an average length of 5.3 µm; it reassociates with a low C0t1/2 after denaturation, and in alkaline isopycnic centrifugation it separates into strands differing in density by 0.005 g/cm3. MtDNA isolated from purified mitochondria of unfertilized eggs or from total larval DNA melts with three distinct thermal transitions. The three melting temperature values suggest that the molecule may have three regions differing in average base composition. DNA isolated from unfertilized eggs of D. melanogaster contains approximately equal amounts of MtDNA and another DNA with a buoyant density of 1.697 g/cm3, slightly less dense than main peak DNA. The possibility that the heavier DNA fraction consists of amplified ribosomal DNA was excluded by hybridization experiments, but otherwise nothing is known of its origin or function. 相似文献
11.
The ratios of satellite deoxyribonucleic acid components to chromosomal deoxyribonucleic acid in Euglena gracilis Z were measured by analytical density gradient ultracentrifugation. Chloroplast deoxyribonucleic acid with a buoyant density of 1.685 g/cm3 exhibited a constant ratio to chromosomal deoxyribonucleic acid during exponential growth and increased twofold as the culture reached the end of the exponential growth phase. The quantity of a satellite deoxyribonucleic acid with a buoyant density of 1.691 g/cm3 was not sufficient to measure the ratio to chromosomal deoxyribonucleic acid during exponential growth but increased to approximately equal the quantity of chloroplast deoxyribonucleic acid as the culture approached the end of the exponential growth phase. The quantity of a deoxyribonucleic acid component with a buoyant density of 1.700 g/cm3 was not sufficient to measure the ratio to chromosomal deoxyribonucleic acid during exponential growth but represented approximately one-third of the total deoxyribonucleic acid as the culture entered the stationary phase of growth. 相似文献
12.
A Cornish-Bowden 《Journal of molecular biology》1976,101(1):1-9
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. 相似文献
13.
Very long runs of pyrimidine nucleotides (polypyrimidines), previously detected in DNA from Drosophila melanogaster, have now been localized to a “cryptic” satellite. These polypyrimidines have an average length of 750 nucleotides and account for about 3% of the thymine residues in total DNA. The buoyant density of the DNA component which contains the polypyrimidines was detected by centrifuging native DNA to equilibrium in a CsCI gradient, and then assaying each fraction for its content of polypyrimidines. A peak was detected at a density of about 1.707 gm/cm3, distinctly heavier than the main band of DNA (1.702 gm/cm3). The buoyant density of polypyrimidine-containing molecules was little affected by differences in the molecular weight of the starting DNA in the range 105-107 daltons (single-stranded). Thus polypyrimidines (and their complementary polypurines) appear to form all or part of a “cryptic” satellite.Polypyrimidines have been isolated and characterized with respect to composition and buoyant density. Direct nucleoside analysis of unlabeled material indicated 34.5% deoxycytidine, 65.5% thymidine. Their banding position in neutral and alkaline CsCI gradients was consistent with a single-stranded DNA polymer of this composition. 相似文献
14.
Amoeba proteus synthesizes DNA in G2 phase of the cell cycle upon feeding after starvation. The characteristics of the DNA synthesized in G2 have been studied by microscope photometry of individual Feulgen-stained nuclei and by buoyant density centrifugation of nuclear DNA in CsCl. Amoeba nuclei were found to contain 42.8 pg of DNA. This DNA bands in CsCl at a density of 1.693 g/cm3 with a satellite at 1.714 g/cm3 which makes up 24% of nuclear DNA. DNA from whole cells has an additional non-nuclear satellite at 1.726 g/cm3. When cells are starved and re-fed with food labeled with [3H]thymidine, the DNA synthesized is predominantly the 1.714 satellite. The amount of DNA synthesized in G2 is small since there is no measurable difference in Feulgen dye binding to nuclei of starved vs starved and re-fed cells. The data suggest that refeeding induces a resumption of late S phase DNA synthesis, or the preferential synthesis of specific DNA sequences such as rRNA genes. 相似文献
15.
The arrangement of the reiterated DNA sequences complementary to transfer RNA has been studied in Xenopus laevis. Prehybridization of denatured DNA with an excess of unfractionated tRNA results in a small but well-defined increase in the buoyant density of fragments which contain sequences homologous to tRNA. The density increase is smaller than that found for 5 S DNA, but is the same or nearly so for all tRNA coding sequences examined. These results indicate that the majority of tRNA genes are clustered together with spacer DNA, the average size of which is estimated to be approximately 0.5 × 106 daltons (native) DNA.In high molecular weight native DNA preparations, the sequences homologous to unfractionated tRNA, tRNAVal, tRNA1Met and tRNA2Met band in CsCl at 1.707, 1.702, 1.708 and 1.711 g cm?3, respectively. The mean buoyant densities are constant at all molecular weights examined but they do not correspond to the base compositions of the complementary tRNA species. These results indicate that isocoding genes are linked to spacer DNA in separate and extensive gene clusters, and that the different clusters contain different spacer DNA sequences. These clusters form well-defined cryptic DNA satellites which are potentially separable from each other as well as from other chromosomal DNA. 相似文献
16.
Ingrid Capesius 《Plant Systematics and Evolution》1979,133(1-2):1-13
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. 相似文献
17.
DNA isolated from purified nuclei of Polytoma obtusum has a buoyant density of 1.711 g/ml in CsCl, a Tm of 91.3° C in SSC, and a G + C content of 52.5% as determined by base composition analysis. Thermal dissociation and reassociation studies indicated that this nuclear DNA contains a considerable amount of heterogeneity. Under appropriate reannealing conditions for denatured DNA, about 15% of the DNA reannealed to form a satellite peak at a density of 1.711 g/ml within one hour. Native DNA fractions of different average buoyant densities, ranging from 1.723 to 1.708 g/ml were also obtained in a preparative CsCl gradient, indicating the presence of intermolecular heterogeneity at a molecular size of 8.5×106 daltons. The nuclear DNA reassociated as three distinct classes. The very fast species constituted about 20 % of the total hyperchromicity, the class of intermediate rate comprised roughly 10% of the nuclear DNA, while the remaining 70% consisted of unique sequences. The haploid genome set was estimated by renaturation kinetics studies to contain 5.0×1010 daltons of DNA or 7.5×107 nucleotide pairs. The analytical complexity of the total nuclear genome was found to be 9.35×1010 daltons, thus indicating that vegetative cells of P. obtusum are diploid. 相似文献
18.
Multiple drug-resistant strains of Pasteurella multocida were associated with a high incidence of fatal pneumonia in feedlot cattle. A representative strain, CAH160, resistant to tetracycline (Tc), streptomycin (Sm), and sulfonamide (Su) was studied. The minimal inhibitory concentration (MIC) of Tc was 32 μg/ml while Sm had an MIC of 256 μg/ml. Plasmid DNA was isolated from CAH160 by cesium chloride-ethidium bromide centrifugation. Agarose gel electrophoresis showed that at least three distinct species of plasmid DNA were present. DNA isolated from CAH160 was used to transform Escherichia coli K12 strain C600 rk?mk?. Transformants resistant to Tc; to Sm, Su; and to Tc, Sm, Su were obtained. Contour length measurements of plasmid DNA isolated from transformant cells showed that Tc resistance was associated with a 3-Mdal plasmid (pSR10), while Sm, Su resistance resided on a 2.7-Mdal molecule (pSR11). More than 20% of the transformants were resistant to Tc, Sm, Su and contained both plasmid species. In E. coli the MIC of Tc was 256 μg/ml and that of Sm was 64 μg/ml. The buoyant density of pSR10 was 1.699 g/cm3, while the density of pSR11 was 1.709 g/cm3. 相似文献
19.
The ribosomal cistrons of the water mold Achlya bisexualis 总被引:2,自引:0,他引:2
Total DNA was extracted from vegatative mycelia of the water mold Achlya bisexualis. Fractionation of the DNA in CsCl gradients resulted in two components: a major component with a buoyant density of 1.697 g cm?3 and a minor component with a density of 1.685 g cm?3. The minor component has been identified as mitochondrial DNA based on extractions from isolated mitochondria and Triton X-100 washed nuclei. Detergent washing of the nuclei yielded DNA in which the mitochondrial DNA component was absent, while the isolated mitochondrial preparations contained DNA enriched in the 1.685 g cm?3 component. Hybridization studies of A. bisexualis DNA to rRNA show that the ribosomal cistrons have a buoyant density coincident with that obtained with the nuclear DNA. In addition, preliminary evidence indicates that the mitochondrial DNA does not hybridize to the cytoplasmic RNA under the conditions used for this study. Ribosomal RNA hybridized to about 0.65% of the total DNA. 相似文献
20.
Summary Circular DNA has been investigated in the water mold Saprolegnia. This organism was shown to contain two satellite DNA components at 1.685 and 1.707 g/cm3 in addition to main band DNA at a buoyant density of 1.717 g/cm3. The component banding at a density of 1.685 g/cm3 was found to occur as closed circles of length 14 m in the relaxed form. On the basis of sucrose gradient studies this DNA is thought to be mitochondrial in origin. No other class of circular molecules was observed. 相似文献