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PubMed1984

Microcomputer programs for back translation of protein to DNA sequences and analysis of ambiguous DNA sequences.

Three computer programs are described which may be used to translate a DNA sequence into a protein sequence, back translate the protein sequence into an ambiguous DNA sequence, and then do pattern searching in the ambiguous sequence. The programs are written in the C programming language, have been compiled to run on a microcomputer under the CP/M 80 operating system, and may be copied in binary format through a modem. They are also to become available for the IBM/PC.

Amino Acid Sequence↗
PubMed1996

Design of sequence-specific DNA binding ligands that use a two-stranded peptide motif for DNA sequence recognition.

The design and DNA binding activity of beta-structure-forming peptides and netropsin-peptide conjugates are reported. It is found that a pair of peptides-S,S'-bis(Lys-Gly-Val-Cys-Val-NH-NH-Dns)-bridged by an S-S bond binds at least 10 times more strongly to poly(dG).poly(dC) than to poly(dA).poly(dT). This peptide can also discriminate between 5'-GpG-3' and 5'-GpC-3' steps in the DNA minor groove. Based on these observations, new synthetic ligands, bis-netropsins, were constructed in which two netropsin-like fragments were attached by means of short linkers to a pair of peptides-Gly-Cys-Gly- or Val-Cys-Val-bridged by S-S bonds. These compounds possess a composite binding specificity: the peptide chains recognize 5'-GpG-3' steps on DNA, whereas the netropsin-like fragments bind preferentially to runs of 4 AT base pairs. Our data indicate that combining the AT-base-pair specific properties of the netropsin-type structure with the 5'-GpG-3'-specific properties of certain oligopeptides offers a new approach to the synthesis of ligands capable of recognizing mixed sequences of AT- and GC-base pairs in the DNA minor groove. These compounds are potential models for DNA-binding domains in proteins which specifically recognize base pair sequences in the minor groove of DNA.

Aminoglycosides↗
PubMed1992

The reverse DNA sequencing using Bst DNA polymerase.

The reverse DNA sequencing (RDS) [1] is a rapid method used to check the DNA sequences by sequencing them from the opposite orientation. Because the RDS is basically a double stranded sequencing, the quality of the sequence patterns so obtained generally is not as good as those obtained by the single stranded sequencing, and extra bands and higher background are produced more frequently. This paper shows that the RDS could now generate as good sequence patterns as those obtained by sequencing on the single stranded DNA template if Bst DNA polymerase instead of the conventional enzymes, such as the Klenow enzyme, was used in the RDS. Bst DNA polymerase is heat stable (optimum reaction temperature 65 degrees C) and has recently been successfully used in the conventional DNA sequencing. The RDS has recently been further simplified to meet the need of large DNA sequencing projects such as the human genome project. The combination of the simplified RDS and the use of Bst polymerase should be expected to facilitate greatly the work on sequence confirmation and correction.

Base Sequence↗
PubMed2006

Single-molecule, motion-based DNA sequencing using RNA polymerase.

We present a method for sequencing DNA that relies on the motion of single RNA polymerase molecules. When a given nucleotide species limits the rate of transcription, polymerase molecules pause at positions corresponding to the rare base. An ultrastable optical trapping apparatus capable of base pair resolution was used to monitor transcription under limiting amounts of each of the four nucleotide species. From the aligned patterns of pauses recorded from as few as four molecules, we determined the DNA sequence. This proof of principle demonstrates that the motion of a processive nucleic acid enzyme may be used to extract sequence information directly from DNA.

Adenosine Triphosphate↗
PubMed1990

DNA sequencing separations in capillary gels on a modified commercial DNA sequencing instrument.

DNA sequencing separations of standard DNA fragments of known sequence have been achieved in small diameter capillary gels electrophoresed and analyzed in parallel in a modified commercial DNA sequencer instrument. DNA sequencing in terms of base-calling accuracy is comparable to conventional slab gels; however, the separations in the capillary were performed somewhat faster and required less sample than those in the slab gel. Advantages of this approach vs. separations on conventional slab gels are discussed.

Base Sequence↗
PubMed1978

pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long.

I have derived a complete restriction map of pBR322 from the total nucleotide sequence of the plasmid. Most of the restriction sites also have been demonstrated empirically. The exact sizes of all restriction fragments and the relative positions of the cuts are presented. These fragments can serve as accurate DNA size markers from small pieces up to the 4362 base pair length of pBR322. Inserts cloned in this vector may be characterized easily using this data.

Base Sequence↗
PubMed1989

Comparative analysis of alternating purine-pyrimidine tracts and potential Z-DNA sequences in DNA plant viruses.

The DNAs of several plant viruses were analyzed for the presence of alternating purine-pyrimidine sequences that can potentially undergo B to Z transition. The DNA of the caulimoviruses (plant retroviruses) was compared with that of the geminiviruses, with the cDNA of an RNA plant virus, and with several computer-generated random sequences. Our analysis indicates that potential Z-DNA sites tend to be restricted in the DNA of the caulimoviruses, whereas the same does not occur significantly in the other viral DNAs examined. This result is discussed in relation to the mode of replication of the caulimoviral DNA and offers additional evidence of the existence of selection processes regulating the frequency and distribution of the Z-DNA sites in the different genomes.

Base Composition↗
PubMed1991

Malignant cell detection in Burkitt's lymphoma using third-complementarity-determining region (CDRIII), clone-specific probe developed by sequencing DNA from stored slides.

The DNA sequence of the third-complementarity-determining region (CDRIII) of the immunoglobulin heavy chain (IgH) gene in a case of Burkitt's lymphoma was determined by polymerase chain reaction (PCR) using template DNA extracted from a smear stored at room temperature for more than one year. The DNA sequence obtained from the stored slide was compared with that of DNA from a frozen lymph node biopsied at the initial presentation. The sequences were shown to be identical, implying that DNA from a smear on a stored slide can be used as a source of DNA for PCR amplification, sequencing, and development of a clone-specific probe. Using oligonucleotides generated from one of the CDRIII sequences of the IgH gene as molecular probes, a retrospective study for the malignant clone on the smears was carried out. Malignant cells were detectable in the peripheral blood at an early stage of bone marrow relapse but not in the peripheral blood or bone marrow at the initial presentation. No malignant clone was detected in the bone marrow when testicular infiltration was diagnosed by examination of a pathological specimen. Thus, the technique permits molecular analysis of hematologic malignancies of B-cell lineage in cases where fresh or frozen specimens are not available.

Base Sequence↗
PubMed1993

Recognition of genes in DNA sequence with ambiguities.

The search for genes in a newly sequenced DNA is a well known problem. Among other factors, the gene-searching process is hampered by a number of ambiguities which may remain unresolved experimentally for a long time. A computer method that is able to predict genes in a DNA sequence containing ambiguities has been developed, based on the non-homogeneous Markov chain technique. The reliability of the method has been tested using a set of sequences generated by a Monte-Carlo procedure and a set of 425 E. coli sequences with ambiguities introduced artificially.

DNA↗
PubMedDate not supplied

[Single copy and repetitive DNA sequences of Echinodermata. I. Arrangement of DNA sequences of different multiplicity].

Arrangement of repetitive and single copy DNA sequences in the DNA of 8 Echinodermata species (sea urchins, starfishes and sea-cucumber) has been studied. Comparison of the reassociation kinetics of short and long DNA fragments assayed by hydroxyapatite binding indicates that the pattern of DNA sequence organization of all these species is similar to the so called Xenopus pattern found in genomes of most animals and plants. Interspecies differences consist mainly in the quantities of sequences of various repetition degrees and their interspersion with each other and with single copy sequences. Measurements of the size of S1 nuclease resistant reassociated repetitive sequences show variability in the relative quantities of long and short repetitive sequences of different species. Difference in the arrangement of single copy and repetitive sequences between Echinodermata species are not related to their evolutionary proximity.

Animals↗
PubMed2004

Detection of inter-spread repeat sequence in genomic DNA sequence.

Various types of periodic patterns in nucleotide sequences are known to be very abundant in a genomic DNA sequence, and to play important biological roles such as gene expression, genome structural stabilization, and recombination. We present a new method, named "STEPSTONE", to find a specific periodic pattern of repeat sequence, inter-spread repeat, in which the tandem repeats of the conserved and the not-conserved regions appear periodically. In our method, at first, the data on periods of short repeat sequences found in a target sequence are stored as a hash data, and then are selected by application of an auto-correlation test in time series analysis. Among the statistically selected sequences, the inter-spread repeats are obtained by usual alignment procedures through two steps. To test the performance of our method, we examined the inter-spread repeats in Mycobacterium tuberculosis and Zamia paucijuga genomic sequences. As a result, our method exactly detected the repeats in the two sequences, being useful for identifying systematically the inter-spread repeats in DNA sequence.

Algorithms↗
PubMed1996

DNA sequencing with [alpha-33P]-labeled ddNTP terminators: a new approach to DNA sequencing with Thermo Sequenase DNA polymerase.

A new approach to DNA sequencing is described. The method is based on the use of [alpha-33P]-labeled dideoxyribonucleoside triphosphate terminators and Thermo Sequenase DNA polymerase in cycle sequencing. Thermo Sequenase DNA polymerase incorporates ddNTPs as efficiently as dNTPs, allowing the use of low concentrations of these nucleotides in DNA sequencing. Because only the properly terminated chains are labeled and visualized on autoradiography of the sequencing gels, the sequence results are free of background. The intensity of DNA bands generated are remarkably uniform, which makes reading of DNA sequences easy. By staggered loading of the sequencing gel (at 2-3 hour intervals), it is possible to sequence DNA at least 450 to 500 nucleotides. Exposure time for autoradiography with [alpha-33P] labels is much shorter than with [35S] and does not substantially compromise autoradiographic resolution. Data can be obtained after only 12 hours of exposure of an X-ray film. Moreover, cycle sequencing requires very small amounts of single- or double-stranded template. Consequently, it is even possible to generate sequence data from a single bacterial colony. The details of the protocol are presented in a stepwise manner, and some important parameters to be considered for sequencing with this method are discussed.

Codon, Terminator↗
PubMed2000

d(GA x TC)(n) microsatellite DNA sequences enhance homologous DNA recombination in SV40 minichromosomes.

The genomic distribution of the abundant eukaryotic d(GA x TC)(n) DNA microsatellite suggests that it could contribute to DNA recombination. Here, it is shown that this type of microsatellite DNA sequence enhances DNA recombination in SV40 minichromosomes, the rate of homologous DNA recombination increasing by as much as two orders of magnitude in the presence of a d(GA x TC)(22) sequence. This effect depends on the region of the SV40 genome at which the d(GA x TC)(22) sequence is cloned. It is high when the sequence is located proximal to the SV40 control region but no effect is observed when located 3.5 kb away from the SV40 ori. These results indicate that the recombination potential of d(GA x TC)(n) sequences is likely linked to DNA replication and/or transcription. The potential contribution of the structural properties of d(GA x TC)(n) sequences to this effect is discussed.

Animals↗
PubMed1976

Endogenous oncornaviral DNA sequences: evidence for two classes of viral DNA sequences in guinea pig cells.

The nature of the endogenous viral DNA sequences in guinea pig cells was studied by hybridization. A segment of the viral RNA (r-VRNA) hybridizing to abundant (or reiterated) DNA sequences (R-VDNA) was isolated by recycling to a Cot of 300. The hybridization of the recycled VRNA, as well as the total VRNA, was followed by determining their kinetics and by Wetmur-Davidson analysis. The kinetics of hybridization of total VRNA were complex, did not follow a second-order kinetics, and revealed two slopes by Wetmur-Davidson analysis. The recycled RNA, on the other hand, had a second-order reaction rate expected of the hybridization between a single species of RNA and DNA sequences and yielded a single straight line in a Wetmur-Davidson plot. The Cot1/2 and slope of the recycled r-VRNA was almost identical to that of the abundant VDNA sequences obtained from the hybridization data of the total VRNA. Guinea pig 28S rRNA with or without recycling was used in monitoring hybridization rate. The kinetics of hybridization of 28S RNA followed a second-order reaction and produced a single straight line by Wetmur-Davidson plot, with a second-order reassociation rate constant of 9.6 x 10(-3) liters/mol-s, a Cot1/2 of 104 mol-s/liter, and reiteration frequency of 146. There was no difference in the kinetics of hybridization of 28S RNA before and after recycling. These experiments showed that guinea pig cells contain two classes of VDNA sequences. (i) R-VDNA sequences with a second-order reassociation rate constant of 8.2 x 10(-4) liters/mol-s, a Cot1/2 of 1,219 mol-s/liter, and a reiteration frequency of 12 represent 37.5% of the viral genome. (ii) Unique VDNA sequences with a second-order reassociation rate constant of 1.2 x 10(-4) liters/mol-s, a Cot1/2 of 7,692 mol-s/liter, and a reiteration frequency of 2 represent 62.5% of the viral genome.

Animals↗