Differences between the endogenous and exogenous DNA sequences of Rous-associated virus-O.

DNA sequences related to the endogenous retrovirus of chickens, Rous-associated virus-O (RAV-O), have been examined using site-specific DNA endonuclease analysis of cellular DNA derived from line 15 and line 100 chickens. Individual embryos from both inbred lines were used as a source of embryonic fibroblasts from which cellular DNA was isolated. Analysis of DNA containing either endogenous RAV-O sequences alone or both endogenous and exogenous RAV-O sequences produced identical patterns of RAV-O-specific DNA fragments after digestion with the endonucleases Eco RI, Hind III, BgI II, Bam HI or Xho I. Similar analysis with endonucleases Hinc II or Hha I, however, produced several RAV-O-specific DNA fragments which were derived from cellular DNA containing both endogenous and exogenous RAV-O sequences but not from cellular DNA containing only endogenous sequences. Although some differences exist between the DNA fragments specific for the endogenous viral sequences of line 15 and line 100 cellular DNA, the DNA fragments specific for the exogenous viral sequences were identical between the two inbred lines. Cleavage of an unintegrated linear RAV-O DNA molecule with Hinc II or Hha I produced DNA fragments identical to those specific for the exogenously acquired RAV-O provirus. This suggests that these characteristic fragments contain no cellular DNA. The potential DNA junction fragments containing both viral and cellular DNA, identified after analysis of DNA that contains both endogenous and exogenous viral sequences, were identical to those observed after analysis of DNA containing only endogenous viral sequences. These results support the following conclusions. First, exogenous proviral sequences are integrated into chicken cell DNA following an interaction between viral and cellular DNA that is specific with respect to the virus and nonspecific with respect to the cell. Second, both the free linear RAV-O DNA intermediate and the newly integrated exogenous provirus contain specific endonuclease sites that are not found in endogenous RAV-O DNA sequences. These results suggest that the formation of the exogenous DNA provirus involves specific alteration of the endogenous viral DNA sequences before reinsertion of the sequences as the exogenous RAV-O DNA provirus. It is possible that newly integrated exogenous RAV-O sequences are characterized by specific differences in the pattern of base methylation and a limited sequence arrangement.     

The study of DNA sequences by their sequences of twist angles

To study the properties of DNA sequences we have transformed the sequences of bases into the sequences of twist angles along the chain of DNA double helix by using the Dickerson sum function. The Fourier transform and the auto-correlation function of the twist angles sequences have been used to study the periodicity and randomness of the original DNA sequences. Basing on the correlation coefficient, a "distance" between two DNA fragments has been defined and used to compare some realistic DNA sequences. It is hoped that the techniques developed here could be used to analyze more realistic DNA sequences.     

Two Dimensional Yau-Hausdorff Distance with Applications on Comparison of DNA and Protein Sequences

Comparing DNA or protein sequences plays an important role in the functional analysis of genomes. Despite many methods available for sequences comparison, few methods retain the information content of sequences. We propose a new approach, the Yau-Hausdorff method, which considers all translations and rotations when seeking the best match of graphical curves of DNA or protein sequences. The complexity of this method is lower than that of any other two dimensional minimum Hausdorff algorithm. The Yau-Hausdorff method can be used for measuring the similarity of DNA sequences based on two important tools: the Yau-Hausdorff distance and graphical representation of DNA sequences. The graphical representations of DNA sequences conserve all sequence information and the Yau-Hausdorff distance is mathematically proved as a true metric. Therefore, the proposed distance can preciously measure the similarity of DNA sequences. The phylogenetic analyses of DNA sequences by the Yau-Hausdorff distance show the accuracy and stability of our approach in similarity comparison of DNA or protein sequences. This study demonstrates that Yau-Hausdorff distance is a natural metric for DNA and protein sequences with high level of stability. The approach can be also applied to similarity analysis of protein sequences by graphic representations, as well as general two dimensional shape matching.     

Sub-set characteristics of DNA sequences involved in tight DNA/polypeptide complexes and their homology to nuclear matrix DNA

Polypeptides co-isolating with DNA induce the binding of a fraction of native DNA fragments to nitrocellulose filters. Southern analysis reveals a high intensity of self-hybridization of the DNA sequences retained on nitrocellulose filters. Consistently, the DNA fraction passing the filters shows only weak hybridization when probed with DNA retained on filters. This indicates that the DNA/polypeptide complexes reside on a non-random sub-set of DNA sequences. Moreover, a high degree of homology was found between residual nuclear matrix DNA sequences and the DNA sequences retained on nitrocellulose filters. This indicates that the DNA sequences associated with tightly bound polypeptides originate from sites where the genome is salt-stably anchored in the nuclear matrix.     

Distribution pattern and enzymic hypermethylation of inverted repetitive DNA sequences in P815 mastocytoma cells.

A specific class of DNA sequences, the inverted repetitive sequences, forms a double-stranded structure within a single linear polynucleotide chain in denatured DNA. The reassociation process is unimolecular and occurs very fast. Quantitative analyses have shown that in mouse P815 cells these sequences comprise about 4% of the nuclear DNA and are interspersed within sequences of other degrees of repetitiveness. After labeling the cells with L-[Me-3H]methionine and [14C]deoxycytidine, relative rates of enzymic DNA methylation were computed on the basis of radioactivities found in pyrimidine residues of the nuclear DNA. The results indicate that in P815 cells, DNA of inverted repetitive sequences is methylated to a level about 50% higher than the normal repetitive DNA sequences and to about 300% higher than the unique and intermediary intermediatry sequences. The biological function of the inverted repetitive sequences, as well as of the role of enzymic methylation of DNA remains unknown.     

SCAR DNA family is enriched in evolutionarily conserved sequences

The family of DNA sequences tightly associated with the synaptonemal complex (SC), or SCAR DNA family, has earlier been described as a specific family of golden hamster genomic DNA sequences. DNA sequences similar to golden hamster SCAR DNA proved to be widespread in the genomes of some vertebrates. A comparison with a sample of random sequences showed that the SCAR DNA family is enriched in evolutionarily conserved sequences, which correlates with the universal SC morphology and processes occurring in meiotic prophase I.     

Analysis of rat repetitive DNA sequences.

Parameters of repetitive sequence organization have been measured in the rat genome. Experiments using melting, hydroxylapatite binding, and single strand specific nuclease digestion have been used to measure the number, length, and arrangement of repeated DNA sequences. Renaturation and melting or S1 nuclease digestion of 1.0 kbp DNA fragment show about 20% of rat DNA sequences are 3000-fold repeated. Renatured duplexes from 4.0 kbp DNA fragments display two repetitive size fractions after nuclease digestion. About 60% of the repeated sequences are 0.2-0.4 kbp long while the remainder are longer than 1.5 kbp. The arrangement of the repeated sequences has been measured by hydroxylapatite fractionation of DNA fragments of varying lengths bearing a repeated sequence. Repeated DNA sequences are interspersed among 2.5 kbp long nonrepeated sequences throughout more than 70% of the rat genome. There are approximately 350 different 3000-fold short repeated sequences in the rat interspersed among 600,000 nonrepeated DNA sequences.     

Simian virus 40 origin auxiliary sequences weakly facilitate T-antigen binding but strongly facilitate DNA unwinding.

The complete simian virus 40 (SV40) origin of DNA replication (ori) consists of a required core sequence flanked by two auxiliary sequences that together increase the rate of DNA replication in monkey cells about 25-fold. Using an extract of SV40-infected monkey cells that reproduced the effects of ori-auxiliary sequences on DNA replication, we examined the ability of ori-auxiliary sequences to facilitate binding of replication factors and to promote DNA unwinding. Although the replicationally active form of T antigen in these extracts had a strong affinity for ori-core, it had only a weak but specific affinity for ori-auxiliary sequences. Deletion of ori-auxiliary sequences reduced the affinity of ori-core for active T antigen by only 1.6-fold, consistent with the fact that saturating concentrations of T antigen in the cell extract did not reduce the stimulatory role of ori-auxiliary sequences in replication. In contrast, deletion of ori-auxiliary sequences reduced the efficiency of ori-specific, T-antigen-dependent DNA unwinding in cell extracts at least 15-fold. With only purified T antigen in the presence of topoisomerase I to unwind purified DNA, ori-auxiliary sequences strongly facilitated T-antigen-dependent DNA conformational changes consistent with melting the first 50 base pairs. Under these conditions, ori-auxiliary sequences had little effect on the binding of T antigen to DNA. Therefore, a primary role of ori-auxiliary sequences in DNA replication is to facilitate T-antigen-dependent DNA unwinding after the T-antigen preinitiation complex is bound to ori-core.     

Characterization of long and short repetitive sequences in the sea urchin genome

Long and short repetitive sequences were purified from the DNA of Paracentrotus lividus under conditions designed to optimize the yield of complete, end to end sequences. Double-stranded long repeat DNA prepared in this manner ranged in length from approximately 3000 to 15 000 nucleotide pairs with average sizes of approximately 6000 base pairs. In the electron microscope, long repeat DNA was observed to possess continuous sequences that often appeared to be terminated by one or more loops and/or fold backs. Long repeat DNA sequences, resheared to 300 base pairs, were found to have an average melting point identical to that for sheared native DNA. Thus, the reassociated duplexes of long repetitive DNA seem to possess very few mismatched base pairs. Reassociation kinetic analyses indicate that the majority of the long repeat sequences are reiterated only 4--7 times per haploid amount of DNA. Melt-reassociation analyses of short repetitive DNA, at several criteria, support the previously held concept that these sequences belong the sets or families of sequences which are inexact copies of one another. Our studies also support hypotheses suggesting that short repetitive sequences belong to families which may have arisen via distinct salttatory events. The relationships between long and short repetitive DNA sequences are considered with respect to widely held concepts of their sequence organization, evolution, and possible functions within eucaryotic genomes. A model for the possible organization of short repeats within long repetitive DNA sequences is also presented.     

transAlign: using amino acids to facilitate the multiple alignment of protein-coding DNA sequences

Background  

Alignments of homologous DNA sequences are crucial for comparative genomics and phylogenetic analysis. However, multiple alignment represents a computationally difficult problem. For protein-coding DNA sequences, it is more advantageous in terms of both speed and accuracy to align the amino-acid sequences specified by the DNA sequences rather than the DNA sequences themselves. Many implementations making use of this concept of "translated alignments" are incomplete in the sense that they require the user to manually translate the DNA sequences and to perform the amino-acid alignment. As such, they are not well suited to large-scale automated alignments of large and/or numerous DNA data sets.     

Structural organization of the genome of the cellular slime mold Dictyostelium discoideum: interspersion of repetitive and single-copy DNA sequences.

The length and interspersion of reiterated and single-copy DNA sequences in Dictyostelium have been examined. The results indicate that approximately 50-60% of the single-copy sequences in DNA fragments 1500 nucleotides long and 75% of the single-copy sequences in fragments 3000 nucleotides long are linked to short interspersed repeat DNA sequences. The average length of these single-copy sequences is 1500 nucleotides. The length of the reiterated DNA has also been analyzed and shows a bimodal distribution. One half is present in sequences greater than 2000 nucleotides long, while the remainder is present as short fragments 250-450 nucleotides long. These shorter fragments are interspersed with the bulk of the single-copy DNA.     

Visual representation of DNA sequences for exon detection using non-parametric spectral estimation techniques

This paper presents a new approach for modeling of DNA sequences for the purpose of exon detection. The proposed model adopts the sum-of-sinusoids concept for the representation of DNA sequences. The objective of the modeling process is to represent the DNA sequence with few coefficients. The modeling process can be performed on the DNA signal as a whole or on a segment-by-segment basis. The created models can be used instead of the original sequences in a further spectral estimation process for exon detection. The accuracy of modeling is evaluated evaluated by using the Root Mean Square Error (RMSE) and the R-square metrics. In addition, non-parametric spectral estimation methods are used for estimating the spectral of both original and modeled DNA sequences. The results of exon detection based on original and modeled DNA sequences coincide to a great extent, which ensures the success of the proposed sum-of-sinusoids method for modeling of DNA sequences.     

Structural genes adjacent to interspersed repetitive DNA sequences

The observation that repetitive and single copy sequences are interspersed in animal DNAs has suggested that repetitive sequences are adjacent to single copy structural gene sequences. To test this concept, single copy DNA sequences contiguous to interspersed repetitive sequences were prepared from sea urchin DNA by hydroxyapatite fractionation (repeat-contiguous DNA fraction). These single copy sequences included about one third of the total nonrepetitive sequence in the genome as determined by the amounts recovered during the hydroxyapatite fractionation and by reassociation kinetics. ³H-labeled mRNA from sea urchin gastrula was prepared by puromycin release from polysomes and used in DNA-driven hybridization reactions. The kinetics of mRNA hybridization reactions with excess whole DNA were carefully measured, and the rate of hybridization was found to be 3–5 times slower than the corresponding single copy DNA driver reassociation rate. The mRNA hybridized with excess repeat-contiguous DNA with similar kinetics relative to the driver DNA. At completion 80% of that mRNA hybridizable with whole DNA (approximately 65%) had reacted with the repeat-contiguous DNA fraction (50%). This result shows that 80–100% of the mRNA molecules present in sea urchin embryos are transcribed from single copy DNA sequences adjacent to interspersed repetitive sequences in the genome.     

DNA sequence organization in the genome of Petroselinum sativum (Umbelliferae)

The genome of parsley was studied by DNA/DNA reassociation to reveal its spectrum of DNA reiteration frequencies and sequence organization. The reassociation of 300 nucleotide DNA fragments indicates the presence of four classes of DNA differing in repetition frequency. These classes are: highly repetitive sequences, fast intermediate repetitive sequences, slow intermediate repetitive sequences, and unique sequences. The repeated classes are reiterated on average 136,000, 3000, and 42 times respectively. A minor part of the genome is made up of palindromes. — The organization of DNA sequences in the P. sativum genome was determined by the reassociation kinetics of DNA fragments of varying length. Further information was derived from S1 nuclease resistance and from hyperchromicity measurements on DNA fragments reassociated to defined C₀t values. — The portion of the genome organized in a short period interspersion pattern amounts to 47%, with the unique sequences on an average 1000 nucleotides long, and most of the repetitive sequences about 300 nucleotides in length, whereas the weight average length may be up to 600 nucleotides. — About 5% unique DNA and 11% slow intermediate repetitive DNA consist of sequences from 10³ up to 10⁴ nucleotides long; these are interspersed with repetitive sequences of unknown length. Long repetitive sequences constitute 33% of the genome, 13% are satellite-like organized, and 20% in long stretches of intermediate repetitive DNA in which highly divergent sequences alternate with sequences that show only minimal divergence. — The results presented indicate remarkable similarities with the genomes of most animal species on which information is available. The most intriguing pecularity of the plant genome derives from its high content of repetitive DNA and the presumed organization of the latter.     

Evolution of DNA binding motifs and operators

A gene regulatory protein with helix-turn-helix (HTH) DNA-binding motif, GalS contains a functional operator within the DNA sequences encoding the HTH region (Nature 369 (1994) 314). We searched for operator-like sequences within the DNA sequences encoding the DNA binding motifs of other regulatory proteins. Five such proteins, DeoR, CytR, LRP, LuxR and PurR, were found to have actual operator or operator-like sequences in the DNA sequences encoding the DNA-binding motif. Except DeoR, all of them including GalS, are known to be auto-regulated. Auto-regulation in case of DeoR has not been investigated. Seven other proteins containing a HTH motif, do not have operator-like sequences in the DNA sequences encoding the HTH motif; none of them, except MerR, are known to be auto-regulated. The DNA binding proteins may have evolved from a common ancestor containing a DNA binding site within its gene segment that encodes the DNA-binding motif to facilitate auto-regulation. We have discussed current evidence for monophyletic or polyphyletic origin of such sequences.     

Maize DNA enrichment by representational difference analysis in a maize chromosome addition line of oat

 The recent recovery of maize (Zea mays L.) single-chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses has provided novel source materials for the potential isolation of maize chromosome-specific sequences for use in genetic mapping and gene cloning. We report here the application of a technique, known as representational difference analysis (RDA), to selectively isolate maize sequences from a maize chromosome-3 addition line of oat. DNA fragments from the addition line and from the oat parent were prepared using BamHI digestion and primer ligation followed by PCR amplification. A subtractive hybridization technique using an excess of the oat parental DNA was employed to reduce the availability for amplification of DNA fragments from the addition lines that were in common with the ones from the oat parental line. After three rounds of hybridization and amplification, the resulting DNA fragments were cloned into a plasmid vector. A DNA library containing 400 clones was constructed by this method. In a test of 18 clones selected at random from this library, four (22%) detected maize-specific repetitive DNA sequences and nine (50%) showed strong hybridization to genomic DNA of maize but weak hybridization to genomic DNA of oat. Among these latter nine clones, three detected low-copy DNA sequences and two of them detected DNA sequences specific to chromosome 3 of maize, the chromosome retained in the source maize addition line of oat. The other eight out of the 13 clones that had strong hybridization to maize DNA detected repetitive DNA sequences or high-copy number sequences present on most or all maize chromosomes. We estimate that the maize DNA sequences were enriched from about 1.8% to over 72% of the total DNA by this procedure. Most of the isolated DNA fragments detected multiple or repeated DNA sequences in maize, indicating that the major part of the maize genome consists of repetitive DNA sequences that do not cross-hybridize to oat genomic sequences. Received: 18 November 1997 / Accepted: 12 March 1998     

Avian retrovirus pp32 DNA binding protein. Preferential binding to the promoter region of long terminal repeat DNA

The avian retrovirus pp32 protein possesses DNA endonuclease activity and unique DNA binding properties. An improved purification procedure was developed for pp32, resulting in a severalfold increase in the yield of this virion protein. By use of the nitrocellulose filter binding assay, the protein retains approximately 2-fold more supercoiled (form I) DNA molecules than equivalent linear duplex DNA molecules. Single-stranded DNA is only slightly preferred over double-stranded DNA for pp32 binding. The pp32 DNA binding sites on form I pBR322 DNA which contained an insert of avian retrovirus long terminal repeat (LTR) DNA were determined. A preformed protein-DNA complex was digested with one of several different multicut restriction enzymes and filtered through nitrocellulose filters. Fragments containing viral LTR DNA sequences and plasmid DNA containing promoter sequences for the ampicillin and tetracycline genes, sequences for the "left-end" inverted repeat of transposon 3, and sequences encompassing the carboxyl terminus of the beta-lactamase gene were preferentially retained on the filter by pp32. Partial mapping of pp32 DNA binding sites on LTR DNA was accomplished by generation of deletions in LTR DNA sequences. The pp32 protein preferentially bound viral DNA fragments which contain the viral promoter (TATTTAA) and the adjacent "R" repeat sequences. Computer analysis revealed that three of the four plasmid DNA fragments retained by pp32 contained LTR DNA promoter-like sequences (one mismatch only) which were part of statistically significant and thermodynamically stable hairpin structures.