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PubMed1979

DNA sequencing and melting curve.

The dependence of DNA absorbance (for light at about 260 nm) on temperature is related to a specific DNA sequence structure in the vicinity of DNA thermal denaturation (the so-called DNA melting or coiling). A straightforward analysis of the experimental DNA melting curve allows us to determine the lengths, the A+T content, and the location in DNA of certain domains. In the case of a specific DNA fragmentation, the order of fragments in DNA can be learned from this analysis, nondestructively and quickly, without fractionating the fragments and other methods of fragmentation. If the DNA nucleotide sequence is known except for some sites and uncertain portions, the analysis determines these sites and the accuracy of the sequence at the portions. This information may complement exact methods of DNA sequencing. The proposed analysis is applied to bacteriophage phiX174, whose melting curve is known. The results are compared to and found to be in an excellent agreement with the known phiX174 nucleotide sequence.

Base Sequence↗
PubMed1976

Effects of X-irradiation on the hybridization of rat thymus nuclear RNA with repeated and unique DNA sequences.

The kinetics of DNA hybridization with heterogeneous nuclear RNA (hnRNA) from normal and phytohaemagglutinin (PHA)-stimulated thymocytes has been studied in control rats and in animals 30 min after exposure to whole-body X-radiation with 400 rad. Irradiation results in a diminished ability of hnRNA to form hybrids with DNA at all C0t values ranging from 10(-3) to 10(4). Since this effect is most pronounced in the regions of low repetitive and unique DNA sequences, it is concluded that whole-body X-irradiation of animals may also suppress the transcription, particularly, in these regions.

Animals↗
PubMed1979

Tumor-specific DNA sequences in human gliomas.

Utilizing the technique of hydroxyapatite chromatography, normal cellular DNAs were used to recycle off the repeat or normal sequences found in [3H]DNA copied off 70S RNA from malignant astrocytomas. The recycled [3H]DNA were then used to hybridize against DNAs from normal human brain tissues and DNAs from malignant astrocytomas or Grade IV astrocytomas. The results indicated the presence of tumor-specific DNA sequences in malignant astrocytomas, absent in normal brain tissues. The percentages were 88% and 7%, respectively. When recycled medulloblastoma 70S[3H]DNA probes were utilized against DNA'S FROM Grade IV astrocytomas and from normal brain, similar results were obtained. The respective percentages of hybridization were 67% and 7%. Thus it would appear that malignant gliomas contain tumor-specific DNA sequences which are not found in normal brain tissues.

Astrocytoma↗
PubMed2024

Use of 3D chaos game representation to quantify DNA sequence similarity with applications for hierarchical clustering.

A 3D chaos game is shown to be a useful way for encoding DNA sequences. Since matching subsequences in DNA converge in space in 3D chaos game encoding, a DNA sequence's 3D chaos game representation can be used to compare DNA sequences without prior alignment and without truncating or padding any of the sequences. Two proposed methods inspired by shape-similarity comparison techniques show that this form of encoding can perform as well as alignment-based techniques for building phylogenetic trees. The first method uses the volume overlap of intersecting spheres and the second uses shape signatures by summarizing the coordinates, oriented angles, and oriented distances of the 3D chaos game trajectory. The methods are tested using: (1) the first exon of the beta-globin gene for 11 species, (2) mitochondrial DNA from four groups of primates, and (3) a set of synthetic DNA sequences. Simulations show that the proposed methods produce distances that reflect the number of mutation events; additionally, on average, distances resulting from deletion mutations are comparable to those produced by substitution mutations.

Animals↗
PubMed1977

A new method for sequencing DNA.

DNA can be sequenced by a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments then identify the positions of that base. We describe reactions that cleave DNA preferentially at guanines, at adenines, at cytosines and thymines equally, and at cytosines alone. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. The technique will permit sequencing of at least 100 bases from the point of labeling.

Adenine↗
PubMed2026

esloco: simulation-based estimation of local coverage in long-read DNA sequencing.

SUMMARY: Long-read DNA sequencing is increasingly applied for whole-genome studies, yet experimental planning often lacks reliable estimates of target region coverage, leading to costly and time-consuming pilot studies and replicates. We present esloco, a Monte Carlo-based simulation framework for estimating local coverage in long-read sequencing experiments, including scenarios with unknown target regions (e.g. viral integration, CRISPR-Cas9) or PCR-free designs (e.g. base modifications). By modeling coverage as a function of sequencing depth and read length distribution, esloco enables informed predictions of local sequencing outcomes. Benchmarking across a 45-gene panel demonstrated close agreement with empirical data, underscoring the framework's reliability. AVAILABILITY AND IMPLEMENTATION: esloco is a Python package available on PyPI (https://pypi.org/project/esloco/), GitHub (https://github.com/aweich/esloco), and Zenodo (https://doi.org/10.5281/zenodo.17776161).

Sequence Analysis, DNA↗
PubMedDate not supplied

Genes and non-coding DNA sequences.

Many new categories of DNA sequence organization in higher organisms have been discovered in the last 20 years. Most of these are not found in bacterial and each in turn is hailed as a likely candidate for those regulatory elements which are assumed to distinguish higher from lower organisms. This article stresses the similarities between their sequence organizations and the continuing lack of convincing function for any of them. It suggests that these sequences often appear after chance recombinational events and their presence then facilitates further change. Within the constraints of proper function the genome is clearly able to find many organizational solutions of apparently equal advantage to the organism. Finally, an example of rapid genome reorganization resulting from selection for resistance to the drug methotrexate is considered.

Animals↗
PubMed1977

Transformation with specific fragments of adenovirus DNAs. II. Analysis of the viral DNA sequences present in cells transformed with a 7% fragment of adenovirus 5 DNA.

Five clones of rat kidney cells transformed by a small restriction endonuclease fragment of adenovirus 5 (Ad5) DNA (fragment HsuI G, which represents the left terminal 7% of the adenovirus genome) were analyzed with respect to the viral DNA sequences present in the cellular DNAs. In these analyses, the kinetics of renaturation of 32P-labeled specific fragments of Ad5 DNA was measured in the presence of a large amount of DNA extracted either from each of the transformed cell lines or from untransformed cells. The fragments were produced by digestion of 32P-labeled adenovirus 5 DNA with endo R.HsuI, or by digestion of 32P-labeled fragment HsuI G of adeno 5 DNA with endo R.HpaI. All five transformed lines were found to contain DNA sequences homologous to 75--80% of Ad5 fragment HsuI G only. Clones II and V contained approximately 48 copies per quantity of diploid cell DNA, clone VI about 35 copies, clone IV 22 copies and clone III 5--10 copies. These results indicate that a viral DNA segment as small as 5.5% of the Ad5 genome, contains sufficient information for the maintenance of transformation.

Adenoviruses, Human↗
PubMed1979

Assembly of the mitochondrial membrane system. The DNA sequence of a mitochondrial ATPase gene in Saccharomyces cerevisiae.

The mitochondrial DNA of a cytoplasmic "petite" mutant (DS400/A3) of Saccharomyces cerevisiae has been characterized by restriction endonuclease analysis and by DNA sequencing. The DNA has a repeat length of 1,800 base pairs and contains the oli 1 and pho 2 loci, two known markers of the ATPase proteolipid subunit. The nucleotide sequence has helped to establish the presence in the DS400/A3 DNA of the proteolipid gene flanked by two long stretches of DNA rich in A + T. The sequence of the structural gene is in excellent agreement with the previously reported primary structure of the proteolipid. The DNA sequence also indicates that the mitochondrial codons of yeast are highly nondegenerate. The proteolipid gene has been precisely localized on the restriction map of the wild type genome. In addition, it has been possible to orient the gene with respect to other genetic markers and to determine the direction of its transcription.

Adenosine Triphosphatases↗
PubMed1978

Detection of specific DNA sequences in yeast by colony hybridization.

A procedure is described for the detection of specific DNA sequences in Saccharomyces cerevisiae. This method allows a rapid screening of a large number of yeast colonies. The yeast cells of each colony, grown on nitrocellulose filters, are converted, in situ, to protoplasts by snail enzyme, and are then lysed and their DNAs are denatured and fixed on the filter. The presence of the specific DNA sequence is detected directly on the filter by hybridization with a radioactive cRNA. We have used successfully this technique to detect the presence or the absence of specific mt DNA sequences in p+, p- and p0 strains, and to detect the presence or the absence of the 2 mum DNA sequences in different strains.

Base Sequence↗
PubMed1978

Mapping of the herpes simplex virus DNA sequences in three herpes simplex virus thymidine kinase-transformed cell lines.

We have made use of a novel filter hybridization approach in order to map the herpes simplex virus (HSV) DNA sequences which are present in three HSV thymidine kinase (TK)-transformed cell lines. The cell line 33A+ which was produced by infection of 3T3 TK- cells with UV-irradiated HSV-2 (333) was found to contain one contiguous stretch of viral DNA sequences which maps between 0.15 and 0.57 on the HSV-2 genome. The sequences mapping from 0.31 to 0.37 were present in 3--4-fold higher abundance than the rest of the viral DNA sequences in this cell line. Cell lines 5A and 8N were produced by transfection of mouse CL1D cells with sheared HSV-1 (1023) DNA. The cell line 5A was found to contain a contiguous set of viral DNA sequences mapping between 0.26 and 0.41 on the HSV-1 genome. The cell line 8N was found to contain three non-contiguous sets of viral DNA sequences, mapping between 0.09 and 0.41, 0.53 and 0.58, and 0.94 and 1.0 on the HSV-1 genome. These results seem to indicate that many different sets of viral DNA sequences can be incorporated into the cell during HSV-mediated biochemical transformation.

Animals↗
PubMed1977

Single-stranded DNA from oncornavirus-infected cells enriched in virus-specific DNA sequences.

We previously found that a minor fraction of single-stranded DNA (ss-DNA) isolated from native nuclear DNA of normal chicken embryonic cells and cells of other species hybridized with bulk nuclear DNA or cellular RNA in great excess. At least one-third of ss-DNA belonging to the nonrepetitious part of the cell genome could be hybridized to homologous RNAs. In the present work, similar results were obtained with ss-DNA from cells of chickens infected by avian myeloblastosis virus (AMV). To investigate whether this enrichment of ss-DNA in transcribed DNA sequences involves provirus DNA, radioactive AMV RNA and cDNA copies of AMV RNA were used. Most of the 70S AMV RNA hybridized much faster to ss-DNA from productively infected leukemic cells than to bulk DNA. cDNA, either double-stranded or single-stranded, made in the presence of actinomycin D hybridized to total nuclear DNA with similar kinetics. In contrast, about half of the double-stranded cDNA molecules hybridized 40-50 times faster to ss-DNA than to total DNA, indicating that only one of the provirus DNA strands seems to be present in ss-DNA. This was confirmed by the fact that relatively insignificant amounts of the ss-cDNA molecules made in the presence of actinomycin D could be annealed to ss-DNA as compared with bulk DNA. These results indicate that actively transcribed DNA sequences can be selectively distributed in the ss-DNA fraction, probably because of single strand breaks in the vicinity of transcription sites.

Animals↗
PubMed1977

DNA sequencing with chain-terminating inhibitors.

A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.

Base Sequence↗
PubMed1979

Studies on DNA sequences in the Osmundaceae.

Phylogenetic relationships of Osmunda cinnamomea, O. claytoniana, and O. regalis were explored by means of DNA sequence comparisons. Hydroxyapatite thermal elution profiles of self-reassociated repetitive DNA fragments were very similar, indicating the absence of gross differences in the amount of recent amplification or addition of repetitive DNA in any of these three genomes. Interspecific DNA sequence comparisons showed, in contrast to our earlier interpretation, that repeated DNA sequences of O. claytoniana are nearly equally diverged from those of O. cinnamomea and O. regalis. Differences between repetitive sequences of the three species can be interpreted as reflecting amplification events which occurred subsequent to speciation. The data obtained suggest that the three Osmunda species most likely arose more or less simultaneously from a common ancestor. These findings were verified in experiments with tracer DNA preparations enriched for single copy sequences. On the basis of the hydridization data presented here and of the fossil record, the rate of single copy sequence divergence in the ferns is comparable to that in the primates, although slower than that observed in other animal taxa. From this first evaluation of rates of DNA evolution in plants it would seem that the rates for plants and animals are roughly comparable. The evidence suggests that species divergence is accompanied by further reiteration of preexisting repeat sequences. The rate of addition of repetitive sequences probably is slower in ferns than in angiosperms. This difference might be attributable to the much larger effective generation time in ferns.

Base Sequence↗
PubMed1979

Simian virus 40-related DNA sequences in a human brain tumor.

Papovaviruses can induce experimental brain neoplasms in animals, and some papovaviruses have been implicated in the formation of various human tumors. We examined a series of seven human brain tumors removed at craniotomy for the presence of viral DNA sequences by the technique of DNA-DNA hybridization. Simian virus 40 (SV40) DNA was labeled in vitro and used as a "probe" for detecting related DNA sequences in cellular DNA extracted from brain tumors. SV40-related DNA sequences were found in DNA extracted from one tumor, a glioblastoma multiforme. It was calculated that approximately 1.2 viral genome equivalents per diploid cell were present in the tumor. Since the rate of reassociation of the probe deviated from ideal second-order kinetics, it is surmised that either only a portion of the SV40 genome was present in tumor cells or, alternatively, that the probe detected a related human papovavirus.

Animals↗
PubMed1977

Infectious linear DNA sequences replicating in simian virus 40-infected cells.

A new class of linear duplex DNA structures that contain simian virus 40 (SV40) DNA sequences and that are replicated during productive infection of cells with SV40 is described. These structures comprise up to 35% of the radioactively labeled DNA molecules that can be isolated by selective extraction. These molecules represent a unique size class corresponding to the length of an open SV40 DNA molecule (FO III), and they contain a heterogeneous population of DNA sequences either of host or of viral origin, as shown by restriction endonuclease analysis and nucleic acid hybridization. Part of the FO III DNA molecules contain viral-host DNA sequences covalently linked with each other. They start to replicate with the onset of SV40 superhelix replication 1 day after infection. Their rate of synthesis is most pronounced 3 days after infection when superhelix replication is already declining. Furthermore, they cannot be chased into other structures. At least a fraction of these molecules is infectious when administered together with DEAE-dextran to permissive cells. After intracellular circularization, superhelical DNA FO I with an aberrant cleavage pattern accumulates. In addition, tumor and viral capsid antigen are induced, and infectious viral progeny is obtained. Infection of cells with purified SV40 FO I DNA does not result in FO III DNA molecules in the infected cells or in the viral progeny. It is suggested, therefore, that these FO III DNA molecules are perpetuated within SV40 virus pools by encapsidation into pseudovirions.

Base Sequence↗
PubMed1977

The evolution of repetitive DNA sequences in sea urchins.

Molecular hybridization of nuclear DNAs has been employed to study the evolution of the repetitive DNA sequences in four species of sea urchin. The data show that relative to S. purpuratus there has been approximately 0.1% sequence divergence per million years in the repetitive DNA sequences of S. droebachiensis, S. franciscanus, and L. pictus. These results confirm that repetitive DNA sequences are strongly conserved during evolution. However, comparison of the extent of base pair mismatch in the repetitive DNA heteroduplexes formed at Cot 20 with those formed at Cot 200 during the hybridization of S. purpuratus and L. pictus DNAs reveals that highly repetitive sequences of sea urchins may diverge more rapidly than do the more moderately repetitive sequences.

Animals↗
PubMed1979

Structure analysis at the ends of the intervening DNA sequences in the chloroplast 23S ribosomal genes of C. reinhardii.

All of the chloroplast 23S ribosomal genes of C. reinhardii are interrupted by a 0.87 kb sequence (Rochaix and Malnoë, 1978). We have sequenced the DNA across the two ends of this intervening element. In parallel, we have examined the nucleotide sequences in the corresponding part of the 23S ribosomal RNA. This allowed us to locate precisely the boundaries between the coding (that is, transcribed into mature 23S rRNA) and the noncoding DNA. The results show that the intervening sequence is flanked by two identical sets of 3 bp (5'-CGT) oriented as direct repeats. In addition, a sequence of 5 bp (5'-CGTGA) lies exactly next to one end and is found very close (16 bp) to the other end, in the coding part of the gene. These two sets are also oriented as direct repeats. Finally, sequences near one end of the intervening element are found with a few alterations near the other end, but in an inverted orientation. Possible interpretations of these results are discussed.

Base Sequence↗