A critical examination of possible fractionations of human DNA according to base composition.

Human DNA has been fractionated according to base composition by sedimentation equilibrium in an HgCl2/Cs2SO4 density gradient, followed by sedimentation equilibrium in an actinomycin/cesium formate density gradient. The fractions of different base composition resulting from this procedure were subsequently analyzed by sedimentation equilibrium in CsCl, DNA renaturation kinetics, and electron microscopy. All fractions contain similar kinetic classes of repeated DNA sequences as judged by renaturation studies. Short (300 nucleotides) interspersed repeated sequences are found in all fractions with no noticeable enrichment for these sequences in any fraction. Repeated sequences from fractions of different base composition are partially able to cross-hybridize, demonstrating that nearly identical repeated sequences occur in molecules of different base composition. These findings are critically compared to reports of successful density gradient fractionations of different human DNA sequence classes.     

加工假基因演化过程中的两种信息流

定义描述DNA序列组分差异性和碱基关联的两个参数,分析了人类加工假基因演化过程中其组分信息和碱基关联信息的变化特征,发现随时间的推移,加工假基因的组分逐步向其侧翼序列漂移,紧邻碱基关联逐步增强。这表明本研究所得参数可很好地用来表征加工假基因的突变信息。     

Structure of DNA near long tandem arrays of alpha satellite DNA at the centromere of human chromosome 7.

The centromeric regions of human chromosomes contain long tracts of tandemly repeated DNA, of which the most extensively characterized is alpha satellite. In a screen for additional centromeric DNA sequences, four phage clones were obtained which contain alpha satellite as well as other sequences not usually found associated with tandemly repeated alpha satellite DNA, including L1 repetitive elements, an Alu element, and a novel AT-rich repeated sequence. The alpha satellite DNA contained within these clones does not demonstrate the higher-order repeat structure typical of tandemly repeated alpha satellite. Two of the clones contain inversions; instead of the usual head-to-tail arrangement of alpha satellite monomers, the direction of the monomers changes partway through each clone. The presence of both inversions was confirmed in human genomic DNA by polymerase chain reaction amplification of the inverted regions. One phage clone contains a junction between alpha satellite DNA and a novel low-copy repeated sequence. The junction between the two types of DNA is abrupt and the junction sequence is characterized by the presence of runs of A's and T's, yielding an overall base composition of 65% AT with local areas > 80% AT. The AT-rich sequence is found in multiple copies on chromosome 7 and homologous sequences are found in (peri)centromeric locations on other human chromosomes, including chromosomes 1, 2, and 16. As such, the AT-rich sequence adjacent to alpha satellite DNA provides a tool for the further study of the DNA from this region of the chromosome. The phage clones examined are located within the same 3.3-Mb SstII restriction fragment on chromosome 7 as the two previously described alpha satellite arrays, D7Z1 and D7Z2. These new clones demonstrate that centromeric repetitive DNA, at least on chromosome 7, may be more heterogeneous in composition and organization than had previously been thought.     

Isolation and characterization of deoxyribonucleic acid from tissue of the woolly mammoth, Mammuthus primigenius

DNA was isolated from tissue samples of several mammoth specimens, radiocarbon dated between 10,000 and 53,000 years old. The DNA was purified by chromatography on hydroxyapatite at 60 degrees C and was characterized as a heterogeneous population of fragments ranging in size from 3000 to 200 base pairs. Thermal denaturation analysis demonstrated that approximately 25% of the DNA had a base composition similar to Asian elephant DNA calculated as 36% G + C. Preliminary analysis by nucleic acid hybridization indicated that only a small fraction of DNA isolated from mammoth tissue (2-5%) was homologous to DNA of Asian elephant, a close living relative of the mammoth. Our results provide the first definitive isolation and characterization of DNA from ancient tissue and suggest a purification strategy that will lead to preparations of DNA from mammoth tissue significantly enriched in elephant-related DNA sequences.     

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.     

Genomic localization of hepatitis B virus in a human hepatoma cell line.

The integration of hepatitis B viral sequences in the human hepatoma Alexander cell line has been investigated after fractionation of the cell line DNA by centrifugation in a Cs2SO4/BAMD (3,6-(bis-acetato mercurimethyl) dioxane) density gradient. Eight out of nine integrated viral sequences were localized in DNA component H3, which only represents 4% of the human genome and matches the base composition of HBV sequences. These results indicate a targeting and/or a higher stability of the latter in a specific, small compartment of the host genome.     

Stochastic models for heterogeneous DNA sequences

The composition of naturally occurring DNA sequences is often strikingly heterogeneous. In this paper, the DNA sequence is viewed as a stochastic process with local compositional properties determined by the states of a hidden Markov chain. The model used is a discrete-state, discreteoutcome version of a general model for non-stationary time series proposed by Kitagawa (1987). A smoothing algorithm is described which can be used to reconstruct the hidden process and produce graphic displays of the compositional structure of a sequence. The problem of parameter estimation is approached using likelihood methods and an EM algorithm for approximating the maximum likelihood estimate is derived. The methods are applied to sequences from yeast mitochondrial DNA, human and mouse mitochondrial DNAs, a human X chromosomal fragment and the complete genome of bacteriophage lambda.     

Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells.

Analysis of the total base composition of DNA from seven different normal human tissues and eight different types of homogeneous human cell populations revealed considerable tissue-specific and cell-specific differences in the extent of methylation of cytosine residues. The two most highly methylated DNAs were from thymus and brain with 1.00 and 0.98 mole percent 5-methylcytosine (m5C), respectively. The two least methylated DNAs from in vivo sources were placental DNA and sperm DNA, which had 0.76 and 0.84 mole percent m5C, respectively. The differences between these two groups of samples were significant with p less than 0.01. The m5C content of DNA from six human cell lines or strains ranged from 0.57 to 0.85 mole percent. The major and minor base composition of DNA fractionated by reassociation kinetics was also determined. The distribution of m5C among these fractions showed little or no variation with tissue or cell type with the possible exception of sperm DNA. In each case, nonrepetitive DNA sequences were hypomethylated compared to unfractionated DNA.     

Constraints upon the composition of supplementary DNA

Speciation in eukaryotes is often accompanied by massive changes in nuclear DNA amount resulting from the accumulation or deletion of DNA base sequences within chromosomes. Our evidence shows that, among related species, the DNA lost or gained during divergence is of a remarkably consistent and restricted composition in respect of the ratio of repetitive to non-repetitive base sequences. It is argued that the restriction may in part at least be imposed by natural selection, in the sense that the only changes tolerable are confined to DNA fractions of particular composition and organisation.     

Application of constant denaturant capillary electrophoresis (CDCE) to mutation detection in humans

Constant denaturant electrophoresis is a DNA separation technique based on the principle of cooperative melting equilibrium. DNA sequences with distinct high and low melting domains can be utilized to separate and identify molecules differing by only one base pair in the lower melting domain. Combined with capillary gel electrophoresis and when coupled with high fidelity DNA amplification, this approach can detect mutants at a fraction of 10⁻⁶. Modifications to the capillary elecctrophoretic system have also increased DNA loading capacity which allows for analysis of rare mutations in a large, heterogeneous population such as DNA samples derived from human tissues. Employment of this technology has determined the first mutational spectrum in human cells and tissues in a mitochondrial sequence without phenotypic selection of mutants.     

Reclassification of Pichia scaptomyzae and Pichia galeiformis

Based on the base composition of nuclear DNA and DNA/DNA hybridization, Pichia galeiformis IFO 10718T was reclassified as a synonym of Pichia manshurica, and Pichia scaptomyzae IFO 1073 1T was confirmed to be a synonym of Pichia membranifaciens. Comparison of 18S rRNA gene sequences indicated that IFO 10731T (P. scaptomyzae) is identical to P. membranifaciens IFO 10215T and IFO 10725, and IFO 10718T (P. galeiformis) is identical to P. manshurica IFO 10726T. These data were consistent with the view that P scaptomyzae and P membranifaciens should be conspecific, as should P. galeiformis and P manshurica. Variation among 26S rRNA gene domain D1/D2 sequences from three P membranifaciens strains indicated that this species encompasses a genetically heterogeneous population.     

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.     

Effect of base composition at the center of inverted repeated DNA sequences on cruciform transitions in DNA

We have analyzed the effect of base composition at the center of symmetry of inverted repeated DNA sequences on cruciform transitions in supercoiled DNA. For this we have constructed two series of palindromic DNA sequences: one set with differing center and one set with differing center and arm sequences. The F series consists of two 96-base pair perfect inverted repeats which are identical except for the central 10 base pairs which consist of pure AT or GC base pairs. The S series was constructed such that the overall base composition of the inverted repeats was identical but in which the positioning of blocks of AT- and GC-rich sequences varied. The rate of cruciform formation for the inverted repeats in plasmid pUC8 was dramatically influenced by the 8-10 base pairs at the center of the inverted repeat. Inverted repeats with 8-10 AT base pairs in the center were kinetically much more active in cruciform formation than inverted repeats with 8-10 GC base pairs in the center. These experiments show a dominant influence of the center sequences of inverted repeats on the rate of cruciform formation.     

Two-dimensional gel analysis of repetitive nuclear DNA sequences in the genome of Physarum polycephalum. Developmental regulation of the ribosomal gene number

The composition of repetitive sequences in restriction patterns of nuclear DNA of Physarum polycephalum was determined by high-resolution gel analysis. Three types of repeated DNA fragments in the size range of (0.2-2) X 10(3) base pairs could be identified as discrete spots on the gels and distinguished by their abundance and above-average base composition of either guanine and cytosine (G + C) or adenine and thymidine (A + T). On comparing the DNA composition from exponentially growing plasmodia with that of starved plasmodia, which have become competent to sporulate and have lost 80% of their nuclei, no change was detected among the (A + T)-rich repeat fractions, whereas several of the (G + C)-rich fractions revealed fewer copies in the DNA prepared from starved cells. As shown by hybridization under saturating conditions, the reduction of several (G + C)-rich repeated sequences in the restricted nuclear DNA in sporulation-competent cells can be explained by a 64% elimination of the extrachromosomal nucleolar ribosomal DNA sequences.     

Protein tyrosine kinase and protein phosphotyrosine phosphatase in normal and psoriatic skin

Either aphidicolin- or thymidine-synchronized human HL-60 cells were used to study the replication pattern of a family of human repetitive DNA sequences, the Eco RI 340 bp family (alpha RI-DNA), and of the ladders of fragments generated in total human DNA after digestion with XbaI and HaeIII (alpha satellite sequences). DNAs replicated in early, middle-early, middle-late and late S periods were labelled with BUdR or with [3H]thymidine. The efficiency of the cell synchronization procedure was confirmed by the transition from a high-GC to a high-AT average base composition of the DNA synthesized going from early to late S periods. By hybridizing EcoRI 340 bp repetitive fragments to BUdR-DNAs it was found that this family of sequences is replicated throughout the entire S period. Comparing fluorograph densitometric scans of [3H]DNAs to the scans of ethidium bromide patterns of total HL-60 DNA digested with XbaI and HaeIII, it was observed that DNA synthesized in different S periods is characterized by approximately the same ladder of fragments, while the intensity of each band may vary through the S phase; in particular, the XbaI 2.4 kb fragment becomes undetectable in late S.     

An overabundance of long oligopurine tracts occurs in the genome of simple and complex eukaryotes.

A search of sequence information in the GenBank files shows that tracts of 15-30 contiguous purines are greatly overrepresented in all eukaryotic species examined, ranging from yeast to human. Such an overabundance does not occur in prokaryotic sequences. The large increase in the number of oligopurine tracts cannot be explained as a simple consequence of base composition, nearest-neighbor frequencies, or the occurrence of an overabundance of oligoadenosine tracts. Oligopurine sequences have previously been shown to be versatile structural elements in DNA, capable of occuring in several alternate conformations. Thus the bias toward long oligopurine tracts in eukaryotic DNA may reflect the usefulness of these structurally versatile sequences in cell function.     

Changes in DNA composition in the evolution of Vicia species

Summary The composition of nuclear DNA in 3 Vicia species are compared. The species V. eriocarpa, V. johannis and V. melanops are from three separate subgeneric sections of Vicia and show a fourfold variation in their amounts of nuclear DNA. DNA melting experiments, buoyant density gradient analysis and Cot reassociation experiments show that the quantitiative change in nuclear DNA between the three species is achieved by changes in the amounts of both repetitive and nonrepetitive DNA sequences. It is suggested that while the increase in the repetitive fraction is achieved by the proliferation of repetitive base sequences the increase in the nonrepetitive fraction is due to the steady accretion of highly diverged base sequences resulting from mutations, deletions, insertions and base sequence rearrangements among families of repetitive sequences.     

Isolation of human sequences that replicate autonomously in human cells.

We have isolated a heterogeneous collection of human genomic sequences which replicate autonomously when introduced into human cells. The novel strategy for the isolation of these sequences involved cloning random human DNA fragments into a defective Epstein-Barr virus vector. This vector alone was unable to replicate in human cells, but appeared to provide for the nuclear retention of linked DNA. The human sequences persist in a long-term replication assay (greater than 2 months) in the presence of the viral nuclear retention sequences. Using a short-term (4-day) assay, we showed that the human sequences are able to replicate in the absence of all viral sequences. The plasmids bearing human sequences were shown to replicate based on the persistence of MboI-sensitive plasmid DNA in the long-term assay and the appearance of DpnI-resistant DNA in the short-term assay. The human sequences were shown to be responsible for the replication activity and may represent authentic human origins of replication.     

Hybridization of polyuridylic acid to human DNA immobilized onto nitrocellulose filters.

The level of deoxyadenylate (da) regions in human DNA was estimated from formation of poly(U)-poly(da) triplexes on nitrocellulose filters that were RNAase resistant. The (dA) rich sequences were determined following mild ribonuclease treatment of the poly(U)-DNA hybrids (5 mug/ml at 25 degreesC for 30 min), where as exhaustive ribonuclease treatment (5 mug/ml at 25 degrees C for 6 hr) estimated the more (dA) pure sequences. The level of (dA) rich regions was 0.39% of the DNA and for the more (dA) pure regions it was 0.07%. The (dA) regions were widely distributed throughout human DNA regardless of base composition or sequence repetition. However, a concentration of (dA) regions into main band CsC1 gradient fractions of DNA and into repeated DNA was observed.     

Introns Form Compositional Clusters in Parallel with the Compositional Clusters of the Coding Sequences to Which they Pertain

This report deals with the study of compositional properties of human gene sequences evaluating similarities and differences among functionally distinct sectors of the gene independently of the reading frame. To retrieve the compositional information of DNA, we present a neighbor base dependent coding system in which the alphabet of 64 letters (DNA triplets) is compressed to an alphabet of 14 letters here termed triplet composons. The triplets containing the same set of distinct bases in whatever order and number form a triplet composon. The reading of the DNA sequence is performed starting at any letter of the initial triplet and then moving, triplet-to-triplet, until the end of the sequence. The readings were made in an overlapping way along the length of the sequences. The analysis of the compositional content in terms of the composon usage frequencies of the gene sequences shows that: (i) the compositional content of the sequences is far from that of random sequences, even in the case of non-protein coding sequences; (ii) coding sequences can be classified as components of compositional clusters; and (iii) intron sequences in a cluster have the same composon usage frequencies, even as their base composition differs notably from that of their home coding sequences. A comparison of the composon usage frequencies between human and mouse homologous genes indicated that two clusters found in humans do not have their counterpart in mouse whereas the others clusters are stable in both species with respect to their composon usage frequencies in both coding and noncoding sequences.