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PubMed2001

Refined localization of twenty-one genes in subregion p13.1 of human chromosome 1.

In this report, we describe a refinement of the human transcript map of chromosome 1p13.1, a subregion undergoing many aberrations in various types of human cancers. Publicly available genetic linkage, radiation hybrid and physical maps, as well as cytogenetic and sequence data were used to establish the relative order and orientation of ten known intragenic markers. The complete sequence of genomic clones of the region, available at the Sanger Centre, provided the tool for further studies performed by BLAST analysis against all cDNA sequences registered in the Genexpress Index2. This allowed us to assign to subband 1p13.1 nine of the ten known genes, an additional member of the gene family of one of these genes and eleven new transcripts. The remaining known gene and one additional new transcript map at the 1p13.1 and 1p13.2 boundary. The corresponding genes may be responsible for disorders related to this region. The resulting transcript map of 1p13.1 is presented in the printed article with additional data available on a dedicated Web site at the address http://idefix.upr420.vjf.cnrs.fr/CARTO.

Chromosome Mapping↗
PubMed1996

Two new mutations (1811 + 1G-->C and Y569C) identified in the CFTR gene in patients of Macedonian and Croatian origin.

Two novel CFTR gene mutations were identified in one patient of the Macedonian and Croatian origin, respectively. The two mutations were detected by the method of denaturing gradient gel electrophoresis (a splicing mutation 1811 + 1G-->C) in intron 11, and by single strand conformation polymorphism analysis (a missence mutation Y569C) in exon 12. The mutations were characterized by direct sequencing of amplified DNA, according to Sanger. These two novel mutations were detected as associated with the delta F508 mutation.

Child, Preschool↗
PubMed2000

[Isolation, identification and certain characteristics of a Hantaan virus proposed as candidate virus for killed vaccine].

OBJECTIVE: Isolation and identification of a Hantaan virus from HFRS patient's serum for killed vaccine development. METHODS: This virus temporally called FT14 detected by IFAT, RT-PCR. Partial sequence of M segment was determined by Sanger' a dideoxy chain termination method. RESULTS: The antigenicity and PCR amplification of the newly isolated virus FT14 proved that it belongs to virus of HTN type. Antiserum to the virus can neutralize many strains of Hantaan virus from different areas. The partial sequence of M segment of FT14, as compared with other HTN strains, showed great differences among nucleotide sequences, while the deduced amino acid sequence is comparatively conservative. The titer of the virus reached its peak at 9 days post inoculation in vitro, then the peak titer could last for a long time. CONCLUSIONS: This virus strain FT14 would be a good candidate virus for killed vaccine development.

Amino Acid Sequence↗
PubMed1997

The Chromosome 6 database at the Sanger Centre.

The Sanger Centre Chromosome 6 Database (6ace) has been developed as the primary means of release of annotated sequencing and mapping information for human chromosome 6 from the Sanger Centre. It is also being used to curate global data from published and unpublished external sources. The rationale behind the development of 6ace is described, together with information as to how to access the database.

Base Sequence↗
PubMed2004

Characterization of synthetic DNA bar codes in Saccharomyces cerevisiae gene-deletion strains.

Incorporation of strain-specific synthetic DNA tags into yeast Saccharomyces cerevisiae gene-deletion strains has enabled identification of gene functions by massively parallel growth rate analysis. However, it is important to confirm the sequences of these tags, because mutations introduced during construction could lead to significant errors in hybridization performance. To validate this experimental system, we sequenced 11,812 synthetic 20-mer molecular bar codes and adjacent sequences (>1.8 megabases synthetic DNA) by pyrosequencing and Sanger methods. At least 31% of the genome-integrated 20-mer tags contain differences from those originally synthesized. However, these mutations result in anomalous hybridization in only a small subset of strains, and the sequence information enables redesign of hybridization probes for arrays. The robust performance of the yeast gene-deletion dual oligonucleotide bar-code design in array hybridization validates the use of molecular bar codes in living cells for tracking their growth phenotype.

DNA Primers↗
PubMed1982

Signal sequence of alkaline phosphatase of Escherichia coli.

The amino acid sequence of the signal sequence of phoA was determined by DNA sequencing by using the dideoxy chain termination technique (Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467, 1977). The template used was single-stranded DNA obtained from M13 on f1 phage derivatives carrying phoA, constructed by in vitro recombination. The results confirm the sequence of the first five amino acids determined by Sarthy et al. (J. Bacteriol. 139:932-939, 1979) and extend the sequence in the same reading frame into the amino terminal region of the mature alkaline phosphatase (Bradshaw et al., Proc. Natl. Acad. Sci. U.S.A., 78:3473-3477, 1981). As was predicted (Inouye and Beckwith, Proc. Natl. Acad. Sci. U.S.A. 74:1440-1444, 1977), the signal sequence was highly hydrophobic. The alteration of DNA sequence was identified for a promoter mutation that results in the expression of phoA independent of the positive control gene phoB and in insensitivity to high phosphate.

Alkaline Phosphatase↗
PubMed1989

Isolation and nucleotide sequence of the hemA gene of Escherichia coli K12.

The hemA gene of Escherichia coli K12 was cloned by complementation of a hemA mutant of this organism. Subcloning of the initial 6.0 kb HindIII fragment allowed the isolation of a 1.5 kb NheI-AvaI fragment which retained the ability to complement the hemA mutant. DNA sequencing by the dideoxy chain terminator method of Sanger showed the presence of an open reading frame (ORF) of 1254 nucleotides, which ends 6 nucleotides beyond the AvaI site. Primer extension experiments showed the existence of a putative transcription initiation site for the hemA gene, located at position 130. A possible promoter sequence was identified upstream from this transcription initiation site, and its functional activity was confirmed by the use of the pK01 promoter-probe vector. Protein synthesis in an in vitro coupled transcription-translation system showed a 46 kDa protein, which corresponds to the mol. wt. of the hemA protein, as deduced from the nucleotide sequence of the gene. No homology was found between the amino acid sequence of the hemA protein of E. coli K12 and known sequences of other delta-aminolevulinic acid synthases (delta-ALAS), suggesting that this protein is different from other delta-ALAS enzymes.

5-Aminolevulinate Synthetase↗
PubMed1986

Nucleotide sequence of the hemC locus encoding porphobilinogen deaminase of Escherichia coli K12.

Porphobilinogen deaminase, the product of the hemC locus in Escherichia coli K12, catalyses the tetrapolymerisation of porphobilinogen (PBG) into the hydroxymethylbilane, preuroporphyrinogen. The hemC locus has been subcloned from the Clarke and Carbon plasmid pLC41-4. The sequence of the hemC structural gene and flanking DNA was determined by the dideoxy chain termination method of Sanger. The structural gene for hemC is located within a 942bp sequence encoding the monomeric PBG deaminase, molecular weight 33,857. The extent of the coding region was confirmed by sequencing the N-terminus of the purified enzyme and by determination of the molecular weight. The hemC locus is closely linked to the cyaA locus, the genes being transcribed in a divergent manner. Upstream of the hemC coding region, a possible promoter and three repeated GGATG sequences were identified. This is the first report of a complete DNA sequence for a structural gene specifying an enzyme of the heme biosynthetic pathway in prokaryotes.

Ammonia-Lyases↗
PubMed2002

In vitro amplification of Toxoplasma gondii rhoptry protein 2 gene and construction of its eukaryotic expression plasmid.

OBJECTIVE: To construct a recombinant eukaryotic expression plasmid containing rhoptry protein 2 (ROP2) gene of Toxoplasma gondii. METHOD: The truncated ROP2 gene was amplified from the genomic DNA of Toxoplasma gondii RH strain and cloned into plasmid pGEX-4T-1. The PCR product was subcloned into an eukaryotic expression plasmid pcDNA3 which was identified by enzyme digestion and PCR amplification. Sequence analysis of the insert in the recombinant plasmid was performed by Sanger's method. RESULTS: The amplified DNA fragment was about 1043 bp in length, with identical sequence with the published ROP2 gene sequence. Enzyme digestion and PCR analysis showed that the truncated ROP2 gene had been successfully cloned into plasmid pGEX-4T-1 and pcDNA3. CONCLUSION: The truncated ROP2 gene of Toxoplasma gondii has been amplified and cloned into the eukaryotic expression plasmid pcDNA3.

Animals↗
PubMed1986

Coliphage M13 cloning system and ddXTP chain-termination method for DNA sequencing.

Two DNA fragments of cytochrome b gene in yeast mitochondrial DNA were sequenced by Messing's M13 cloning system and Sanger's ddXTP chain-termination method. M13mp8 and M13mp9 serve as vectors for insert fragment. The recipient strain is E. coli JM103 for transfection. When the ratio of insert/vector was 3:1, high frequencies of recombination and positive recombination were obtained. The two fragments, which have 575 bp and 709 bp, were sequenced. To read more bases, the ratio of ddXTP/dXTP must be reduced. On one X-ray film of 80 x 17 cm 394 bp were read.

Base Sequence↗
PubMed2000

Genomics - the new rock and roll?

The end of the beginning of the Human Genome Project was announced on 26 June when the working draft or first assembly was announced. Here, Ian Dunham who led the group at the Sanger Centre that produced the first complete sequence of a human chromosome reflects on how it felt to be with the genome project from the beginning.

Chromosomes, Human, Pair 22↗
PubMed1978

Direct comparison of theoretical and experimental melting profiles for RF II phiX174 DNA.

The determination by Sanger et al. of the comple nucleotide sequence for phiX174 DNA has made it possible for the first time to compare directly theoretical and experimental DNA melting profiles. The comparison shows that the theory predicts the observed shape of the differential melting curve surprisingly well. Calculation of the denaturation maps allows the peaks on the curve to be correlated with cooperative melting out of concrete regions on the sequence of nucleotides.

Base Sequence↗
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↗