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PubMed1978

Complete sequence of constant and 3' noncoding regions of an immunoglobulin mRNA using the dideoxynucleotide method of RNA sequencing.

Three synthetic oligonucleotides were prepared to be complementary to known regions of the mouse immunoglublin light chain mRNA, and their ability to prime the transcription of complementary DNA (cDNA) was studied. The sequence of the cDNA was determined by adapting for mRNA the DNA sequencing method of Sanger, Nicklen and Coulson (1977) which uses 2'3' dideoxy ribonucleotides. A continuous sequence of 532 nucleotides was obtained, 321 corresponding to the whole of the constant region of the mRNA and the remaining 211 being the complete 3' noncoding region of the mRNA. The termination codon U-A-G occurs at the expected position in the mRNA corresponding to the triplet following the C terminal cystine. The nucleotide sequence is partially corroborated by the sequence of fragments obtained previously from 32P-mRNA fingerprints and endonuclease IV digests of 32P-cDNA, and is in agreement with the amino acid sequence of the constant region, except for a rearrangement of four amino acids (between amino acid positions 163 and 166). A revision of the amino acid sequence confirms the nucleic acid sequence.

Amino Acid Sequence↗
PubMed1991

Applications of mass spectrometry to DNA sequencing.

The ability of the mass spectrometer to analyze collectively the masses of DNA fragments that are produced in the Sanger procedure for sequencing may allow the gel electrophoresis step to be eliminated. On the other hand, if gel electrophoresis is required, the use of resonance ionization spectroscopy coupled to a mass spectrometer may enable much faster analysis of DNA bands labeled with stable isotopes. Other combinations of labeling of the DNA and its mass spectrometric analysis with or without gel electrophoresis are also considered. Recent advances in these areas of mass spectrometry are reviewed.

Base Sequence↗
PubMed2000

Autoregulated expression of Schizosaccharomyces pombe meiosis-specific transcription factor Mei4 and a genome-wide search for its target genes.

The Schizosaccharomyces pombe mei4(+) gene encoding a forkhead transcription factor is necessary for the progression of meiosis and sporulation. We searched for novel meiotic genes, the expression of which is dependent on Mei4p, since only the spo6(+) gene has been assigned to its targets. Six known genes responsible for meiotic recombination were examined by Northern blotting, but none were Mei4 dependent for transcription. We determined the important cis-acting element, designated FLEX, to which Mei4p can bind. The S. pombe genome sequence database (The Sanger Centre, UK) was scanned for the central core heptamer and its flanking 3' sequence of FLEX composed of 17 nucleotides, and 10 candidate targets of Mei4 were selected. These contained a FLEX-like sequence in the 5' upstream nontranslatable region within 1 kb of the initiation codon. Northern blotting confirmed that 9 of them, named mde1(+) to mde9(+), were transcriptionally induced during meiosis and were dependent on mei4(+). Most mde genes have not been genetically defined yet, except for mde9(+), which is identical to spn5(+), which encodes one of the septin family of proteins. mde3(+) and a related gene pit1(+) encode proteins related to Saccharomyces cerevisiae Ime2. The double disruptant frequently produced asci having an abnormal number and size of spores, although it completed meiosis. We also found that the forkhead DNA-binding domain of Mei4p binds to the FLEX-like element in the putative promoter region of mei4 and that the maximum induction level of mei4 mRNA required functional mei4 activity. Furthermore, expression of a reporter gene driven by the authentic mei4 promoter was induced in vegetative cells by ectopic overproduction of Mei4p. These results suggest that mei4 transcription is positively autoregulated.

Amino Acid Sequence↗
PubMed2001

[rDNA its sequencing of Herba Dendrobii (Huangcao)].

AIM: To study the genetic diversity of ITS sequences of Herba Dendrobii (Huangcao) and analyze the utility of ITS sequences in molecular authentication of Herba Dendrobii (Huangcao) and phylogenetic of Dendrobium. METHODS: The ITS gene fragment was amplified using a pair of primers. The PCR products were purified and sequenced by the methods of Sanger Dideoxy. RESULTS: The DNA sequence of 228-233 bp ITS 1, 242-247 bp ITS 2 gene fragments and 5.8S rDNA were obtained from 14 samples of Dendrobium. The interspecific substitution varies from 11.79% to 31.58% at ITS 1 and 10.29% to 25.30% at ITS 2. The intraspecific substitution of D. nobile is 0.87% at ITS 1 and without difference at ITS 2. The substitution between Dendrobium and outgroup varies from 23.56% to 36.89% at ITS 1 and 26.52% to 33.31% at ITS 2. The phylogenetic tree based on ITS 1 and ITS 2 data was set up. CONCLUSION: The ITS 1 and ITS 2 gene fragments were highly conservative at intraspecific level in Dendrebium, while they were less conservative at interspecific level in D. nobile. They were least conservative between Dendrobium and outgroup. Hence, the sequence of this fragment is a good molecular marker for authentication of the Huangcao. But, further study is necessary for the phylogenetic of Dendrobium.

Base Sequence↗
PubMedDate not supplied

Synthetic oligodeoxyribonucleotides as tools in molecular genetics: the characterization of the CYC1 (iso-1-cytochrome c encoding) locus of Saccharomyces cerevisiae.

The use of synthetic oligodeoxyribonucleotides as tools for the isolation, characterization and mutagenesis of eukaryote genes has played a major role in the molecular definition of the CYC1 locus of Saccharomyces cerevisiae, which is the structural gene for the apoprotein of iso-1-cytochrome c. Thus, the possibility of using a synthetic oligodeoxyribonucleotide as a probe to identify and monitor the isolation of a specific gene was first established by model studies which defined the melting temperatures (TmS) for duplexes of oligonucleotides of different lengths and base compositions. This led to the isolation of the CYC1 locus using a synthetic 13 nucleotide probe. A more convenient strategy for determination of DNA sequences by the Sanger method was provided by using synthetic oligodeoxyribonucleotides as primers with denatured double-strand plasmid DNA as template. By this means, the sequence of the CYC1 locus was determined by "walking" along the gene without isolating restriction fragments of the DNA or separating DNA strands. Synthetic oligodeoxyribonucleotides, used as primers for reverse transcriptase with mRNA as template, were also used to precisely define the 5'- and 3'-ends of the iso-1-cytochrome c mRNA. Yet another application of synthetic oligodeoxyribonucleotides is their use as specific mutagens after in vitro incorporation into double-stranded DNA. In the case of iso-1-cytochrome c, this mutagenic strategy is being used to define the role of conserved amino-acids in cytochrome function.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Mapping↗
PubMed1979

The use of exonuclease III for preparing single stranded DNA for use as a template in the chain terminator sequencing method.

Exonuclease III, which degrades DNA 3' leads to 5' and is specific for duplex DNA, can be used to prepare single stranded DNA from linear duplexes. This is shown to be suitable as a template for use with the chain terminator DNA sequencing method of Sanger et al. [1]. Strategies are discussed for preparing single stranded DNA templates by this method and, in particular, its application to the sequence analysis of DNA cloned in plasmid vectors.

Base Sequence↗
PubMedDate not supplied

[Economical sequencing of DNA with terminators].

We describe several improvements of chain-termination DNA sequencing procedure of Sanger et al. For template preparation we use 0.3 ml cultures of M13 clones, grown in standard 1,5 ml polypropylene tubes. The sequencing experiment differs from the previously described by the use of deoxyNTP, labelled with phosphorus-33 (a low energy isotope with a half-life of 25 days, commercially produced in the USSR), and by a "quasi-end labelling" reaction, preceding the DNA synthesis in the presence of dideoxyNTPs. The combination of the phosphorus-33 and the quasi-end labelling produces very sharp sequencing ladders, that equal or exceed in quality those obtained with sulphur-35, and only an overnight exposure with a conventional X-ray film is required. The use of plastic tubes for bacterial growth and the 60-well microchambers for carrying out sequencing reactions results in substantial saving of time and cost in routine "middle scale" sequencing (both types of plasticware are produced in the USSR).

Cloning, Molecular↗
PubMed1996

DNA sequencing by delayed extraction-matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a delayed ion extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40- and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.

Base Sequence↗
PubMed2006

What is the future of electrophoresis in large-scale genomic sequencing?

Although a finished human genome reference sequence is now available, the ability to sequence large, complex genomes remains critically important for researchers in the biological sciences, and in particular, continued human genomic sequence determination will ultimately help to realize the promise of medical care tailored to an individual's unique genetic identity. Many new technologies are being developed to decrease the costs and to dramatically increase the data acquisition rate of such sequencing projects. These new sequencing approaches include Sanger reaction-based technologies that have electrophoresis as the final separation step as well as those that use completely novel, nonelectrophoretic methods to generate sequence data. In this review, we discuss the various advances in sequencing technologies and evaluate the current limitations of novel methods that currently preclude their complete acceptance in large-scale sequencing projects. Our primary goal is to analyze and predict the continuing role of electrophoresis in large-scale DNA sequencing, both in the near and longer term.

Animals↗
PubMed1992

Molecular cloning and characterization of the structural gene coding for the developmentally regulated lysosomal enzyme, alpha-mannosidase, in Dictyostelium discoideum.

The gene coding for the Dictyostelium discoideum lysosomal enzyme, alpha-mannosidase, has been cloned and sequenced. To accomplish this, the mature 60- and 58-kDa subunits of the enzyme were purified and subjected to liquid-phase N-terminal amino acid sequencing. Sequence information was obtained for both of the mature subunits, and a 48-mer oligonucleotide was synthesized based on the determined amino acid sequence of the 58-kDa subunit. Using this oligonucleotide as a probe, an 8-kilobase HindIII fragment of genomic DNA was isolated and subjected to Sanger dideoxy DNA sequencing. The first 4400 nucleotides contained the complete alpha-mannosidase gene and 1100 nucleotides of 5'-flanking DNA. Primer extension analysis indicated that transcription begins at multiple sites -48 to -64 nucleotides upstream of the first nucleotide of the predicted translation initiation codon. A single open reading frame (ORF) of 3015 nucleotides was found that was interrupted by a single intron and that contained the amino acid sequences of the N termini of the two mature alpha-mannosidase subunits; a polyadenylation signal was also found just downstream of the termination codon. A potential cleavable signal sequence was identified in the first 22 amino acids of the predicted precursor protein, and two propeptide regions (Pro I and II) were identified that were immediately upstream of the N termini of the 60- and 58-kDa mature subunits, respectively. These propeptide regions are not present in the mature protein and are therefore predicted to be proteolytically removed as the membrane associated 140-kDa precursor is transported to lysosomes and processed to the soluble 60- and 58-kDa mature forms of the enzyme. In fact, potential proteolytic cleavage sites were identified flanking the Pro I and Pro II regions. Pro I, which immediately follows the signal sequence, consists of 18 amino acids, most of which are highly charged and hydrophilic residues, while Pro II, found in the central portion of the precursor, is very hydrophobic. While no obvious transmembrane regions were identified, several short hydrophobic amino acid stretches were found to be localized in and around the Pro II region, and these may be responsible for attachment of precursors to membranes.

Amino Acid Sequence↗
PubMed1981

Sequence of DNA complementary to a small RNA segment of influenza virus A/NT/60/68.

A small RNA segment from the influenza virus strain A/NT/60/68 (H3N2) was converted to cDNA and then to double-stranded DNA using synthetic oligodeoxynucleotide primers. The double-stranded form was cloned into the bacteriophage M1 3mp7. Clones yielding single-strand recombinant templates in opposite orientation were sequenced by the Sanger dideoxynucleotide chain termination technique. The small viral RNA was 422 nucleotides long and the evidence indicated that it was formed by internal deletion of segment 3. It also contained sequences homologous to segment 1.

Base Sequence↗
PubMed2004

GeneDB: a resource for prokaryotic and eukaryotic organisms.

GeneDB (http://www.genedb.org/) is a genome database for prokaryotic and eukaryotic organisms. The resource provides a portal through which data generated by the Pathogen Sequencing Unit at the Wellcome Trust Sanger Institute and other collaborating sequencing centres can be made publicly available. It combines data from finished and ongoing genome and expressed sequence tag (EST) projects with curated annotation, that can be searched, sorted and downloaded, using a single web based resource. The current release stores 11 datasets of which six are curated and maintained by biologists, who review and incorporate information from the scientific literature, public databases and the respective research communities.

Animals↗
PubMed1983

Anti-oxazolone hybridomas and the structure of the oxazolone idiotype.

Antibodies raised in several mouse and rat strains against the hapten 2-phenyloxazolone (phOx, "oxazolone") regularly contain a fraction recognized by antiidiotypic reagents. We have studied this response in BALB/c and DBA/2 mice by generating over fifty anti-phOx antibody-secreting hybridoma clones. The hybridization was performed either 7 or 14 days after a primary immunization with phOx-protein conjugate. Most of the hybrids secreted IgG1. Whereas over 80% (17/21) of IgG-producing hybrids from day-7 fusions secreted oxazolone-idiotype positive immunoglobulin, all hybridomas originating from day-14 fusions were idiotype negative. The mRNA for heavy (H) and light (L) chains of three idiotype-positive and one idiotype-negative IgG1 hybridomas were sequenced by a modification of Sanger's dideoxynucleotide method of DNA sequencing, using crude mRNA as template, synthetic oligonucleotides as primers, and reverse transcriptase to incorporate both dideoxynucleotides and labeled deoxynucleotides. The sequence of the mRNA coding for the whole variable region of each chain was established using primers complementary to the constant region near the V-C boundary and another two that coded for a framework segment in either VH or VL. This method not only provided more information than protein sequencing but was also faster and simpler. The mRNA preparation did not need fractionation beyond the poly A-containing fraction. The sequences of the H and L chain mRNA of the three idiotype-positive anti-oxazolone antibodies were extremely similar or identical, and from them a tentative oxazolone-idiotype basic sequence was derived. Only three nucleotide differences were detected; these occurred in the D segment of one H chain mRNA, in the V-J boundary of one of the light chain mRNA, and in the first hypervariable region of another. The idiotype-negative antibody had a totally different H chain mRNA and a light chain mRNA that differed by 21 bases, almost all affecting the amino acid sequence.

Amino Acid Sequence↗
PubMed1998

[Cloning and sequencing of cDNA in NS3 region of Chinese hepatitis G virus isolated from Henan Province].

To study the genetic characteristics of non-structural region 3 of HGV in China, a HGV-RNA positive sample was collected from Henan. HGV RNA was extracted and amplified by RT-nested-PCR. We got four overlap fragments and they were cloned into plasmid vector pcDNA II. The cDNA was sequenced by the Sanger's method. Nucleotide homology between Henan strain and other reported HGV isolates (PNF2161, R10291, GBV-C) was 85.7% to 93.8% over the 2137 nucleotides examined. Comparison of amino acid sequence deduced from Henan strain with that of other isolates showed 96.1% to 97.7% homology. Three amino acid variations were found in NS3 region of all the three Chinese isolates of HGV. In addition, a N-glycosylation site was found in the NS3 region of all the six isolates.

Amino Acid Sequence↗
PubMed1994

Direct sequencing of PCR-amplified DNA.

This article describes the direct sequencing of PCR-amplified DNA, a technique that bypasses the problem of replication errors sometimes associated with other PCR procedures. The direct sequencing procedure produces an "average sequence" of all the copies of the target. Any miscopied molecule usually represents only a small proportion of the total. The technique described here is based on the "traditional" ddNTP sequencing method of Sanger et al.

Autoradiography↗
PubMedDate not supplied

Complement factor H: sequence analysis of 221 kb of human genomic DNA containing the entire fH, fHR-1 and fHR-3 genes.

Complement factor H (fH) is a member of a family of proteins involved in the regulation of complement activation (RCA). These proteins share a common structural motif, the Short Consensus Repeat (SCR), which is structurally conserved among related genes and between phylogenetically divergent species. fH is composed of 20 such SCRs and a variety of biological functions have been localised to specific SCR domains. The majority of individual SCRs identified are encoded by single exons, and processes such as gene conversion, duplication and exon shuffling have been implicated in the evolution and genomic radiation of SCR-encoding genes. We have analysed two GenBank sequence entries relating to two overlapping PAC clones sequenced at the Sanger Centre which contain the entire human fH gene and two adjacent fH-related (fHR) genes, fHR-1 and fHR-3. Here, we report the detailed analysis of the assembled 221 kb of contiguous, ungapped genomic sequence from human chromosome 1q32, in part employing the RUMMAGE-DP automated annotation tool. Genomic duplications involving fH and fHR exons were identified and Alu/L1 repeat dating established that the duplications occurred after the separation of rodent and primate lineages. The analysis indicates that retrotransposition as well as single and multiple exon duplication events are likely to have been involved in SCR radiation and RCA gene evolution, facilitated by conservation of splice-phasing and the single-exon, single-SCR nature of the encoded domains.

5' Untranslated Regions↗
PubMed1989

[Gene technology in clinical medicine--DNA sequencing].

There are two established procedures for sequencing cloned DNA fragments. In Maxam-Gilbert sequencing, or sequencing by chemical degradation, end-labeled DNA is treated with various chemicals which induce base specific chain breaks with a low frequency. In dideoxy sequencing, or sequencing by the Sanger procedure, a single-stranded DNA is used as a template for synthesis of a labeled complementary strand by a DNA polymerase. Addition of dideoxynucleoside triphosphates will induce base-specific chain termination. In both procedures the nucleotide sequence of the cloned DNA can be deduced after fractionation of the labeled products by polyacrylamide gel electrophoresis. As yet, radioactive labeling of the reaction products is most common, but fluorescence labeling and computer-assisted automated sequence interpretation has become as a powerful alternative during the last years. Further automation of the various processes involved in DNA sequencing will be necessary for the planned sequencing of large genomes, such as the Escherichia coli genome, the yeast genome, and the human genome.

Animals↗