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PubMed1999

Identification and characterization of TspA, a major CD4(+) T-cell- and B-cell-stimulating Neisseria-specific antigen.

In search for novel T-cell immunogens involved in protection against invasive meningococcal disease, we screened fractionated proteins of Neisseria meningitidis (strain SD, B:15:P1.16) by using peripheral blood mononuclear cells (PBMCs) and specific T-cell lines obtained from normal individuals and patients convalescing from N. meningitidis infection. Proteins of iron-depleted meningococci produced higher PBMC proliferation indices than proteins of iron-replete organisms, indicating that iron-regulated proteins are T-cell immunogens. Insoluble proteins of the iron-depleted cells, which produced better T-cell stimulation than soluble ones, were fractionated by using sodium dodecyl sulfate-polyacrylamide gels and recovered as five fractions (F1 to F5) corresponding to decreasing molecular weight ranges. The proteins were purified (by elution and precipitation) or electroblotted onto nitrocellulose membranes (dissolved and precipitated) before use in further T-cell proliferation assays. One of the fractions (F1), containing high-molecular-mass proteins (>130 kDa), consistently showed the strongest T-cell proliferation responses in all of the T-cell lines examined. F1 proteins were subdivided into four smaller fractions (F1A to F1D) which were reexamined in T-cell proliferation assays, and F1C induced the strongest responses in patients' T-cell lines. Rabbit polyclonal antibodies to F1C components were used to screen a genomic expression library of N. meningitidis. Two major clones (C1 and C24) of recombinant meningococcal DNA were identified and fully sequenced. Sequence analysis showed that C24 (1,874 bp) consisted of a single open reading frame (ORF), which was included in clone C1 (2, 778 bp). The strong CD4(+) T-cell-stimulating effect of the polypeptide product of this ORF (named TspA) was confirmed, using a patient T-cell line. Immunogenicity for B cells was confirmed by showing that convalescent patients' serum antibodies recognized TspA on Western blots. Additional genetic sequence downstream of C24 was obtained from the meningococcal genomic sequence database (Sanger Centre), enabling the whole gene of 2,761 bp to be reconstructed. The DNA and deduced amino acid sequence data for tspA failed to show significant homology to any known gene, except for a corresponding (uncharacterized) gene in Neisseria gonorrhoeae genome sequences, suggesting that tspA is unique to the genus Neisseria. The DNA and deduced amino acid sequence of the second ORF of clone C1 showed significant homology to gloA, encoding glyoxalase I enzyme, of Salmonella typhimurium and Escherichia coli. Thus, we have identified a novel neisserial protein (TspA) which proved to be a strong CD4(+) T-cell- and B-cell-stimulating immunogen with potential as a possible vaccine candidate.

Animals↗
PubMed1999

[Cloning and sequencing of the genes coding for the histidine-rich protein II of Plasmodium falciparum].

AIM: To compare and analyze the homology of genes encoding histidine-rich proteinII (HRPII) of different Plasmodium falciparum isolates. METHODS: Using PCR technique, the complete genes coding for HRPII of P. falciparum isolates FCC1/HN and VN isolates were amplified. PCR products were digested by HindIII/BamHI and cloned into plasmid pUC19. The recombinant plasmid HRPII/pUC19 was screened and identified by PCR and restriction analysis. The cloned HRPII genes were sequenced by Sanger's method. RESULTS: HRPII genes of FCC1/HN and VN isolates were successfully amplified and cloned into pUC19. DNA sequencing showed that the coding length of HRPII gene was 1,020 bp without introns in FCC1/HN and VN isolates, however, there were ten points mutations between them. FCC1/HN isolate exhibited 98.8%, 92.2% and 98.7% homology in amino acids with isolates VN, IMTM22, and Itg2, respectively. Though the numbers of repeat sequences were different in four isolates, they had the same hydrophobic leader sequence and a single putative glycosylation site. The secondary structure analysis showed that the main antigenic determinants of four isolates were located on 5' end non-repeat region (amino acids 1-60). CONCLUSION: FCC1/HN isolate was highly homologous in the coding region of HRPII with VN, IMTM22, and Itg2 isolate. Four isolates exhibited similar structural characteristics and antigenic determinants in HRPII.

Amino Acid Sequence↗
PubMedDate not supplied

Quick screening of plasmid deletion clones carrying inserts of desired sizes for DNA sequencing.

Plasmids pWQX001 and pWQX005, constructed from pGEM-4 with an insert of 6.5 kb, were unidirectionally digested with exonuclease III and exonuclease VII. The DNA digests were ligated and used to transform competent cells of Escherichia coli DH5-alpha. The size of the deletion plasmid carried by each transformant was estimated through agarose gel electrophoresis of crude lysates without any purification of the plasmid DNA. Colonies carrying plasmid DNAs with different deletions of the insert were grown and their DNAs were purified through a miniprocedure. The size of each purified plasmid DNA was determined accurately after linearization of the plasmid with an appropriate restriction endonuclease. The remainder of the DNA preparation was sufficiently pure to be sequenced using Sanger's dideoxynucleotide chain termination method. An easy, quick procedure is described for the preliminary selection of templates for DNA sequencing after construction of deletion clones of recombinant plasmid DNA using exonuclease III and exonuclease VII. This procedure permits a rapid screening of large numbers of colonies and selection of those carrying plasmid DNAs with inserts of the desired sizes for sequencing. This procedure does not require purification of the deletion plasmid DNA.

Base Sequence↗
PubMed1992

DNA sequencing using capillary array electrophoresis.

A DNA sequencing method is presented that utilizes capillary array electrophoresis, two-color fluorescence detection, and a two-dye labeling protocol. Sanger DNA sequencing fragments are separated on an array of capillaries and detected on-column using a two-color, laser-excited, confocal-fluorescence scanner. The four sets of DNA sequencing fragments are separated in a single capillary and then distinguished by using a binary coding scheme where each fragment set is labeled with a characteristic ratio of two dye-labeled primers. Since only two dye-labeled primers are required, it is possible to select dyes that have identical mobility shifts. It is also shown that the ratio of the signal in the two detection channels provides a reliable identification of the sequencing fragment. DNA sequencing results on a 25-capillary array are presented.

Base Sequence↗
PubMed2004

Molecular typing of Neisseria gonorrhoeae isolates by pyrosequencing of highly polymorphic segments of the porB gene.

For prevention and control of gonorrhea, an objective, highly discriminating, and reproducible molecular epidemiological characterization of Neisseria gonorrhoeae is essential. In the present study, in pursuance of providing such qualities, pyrosequencing technology, a fast real-time DNA sequence analysis, was applied to six short, highly polymorphic porB gene segments, with subsequent genetic variant (genovar) determination of the bacterial isolates. The sequencing templates were obtained by real-time PCR amplification, which also included fluorescence melting curve analysis of the entire porB gene in order to determine the genogroup (porB1a or porB1b allele) prior to pyrosequencing analysis. The PSQ 96 MA system used allowed rapid (in approximately 1.5 h) determination of 96 sequences of 20 to 65 correct nucleotides each. The results were reproducible and mostly in concordance with the results of conventional Sanger dideoxy sequencing, with the exception of shorter read lengths and some uncertainty in determining the correct number of identical nucleotides in homopolymeric segments. The number of sequence variants identified in each of the six highly polymorphic segments of the porB1a and porB1b alleles (encoding surface-exposed amino acid loops of the mature PorB protein) ranged from 5 to 11 and from 8 to 39, respectively. Among porB1a isolates (n = 22) and porB1b isolates (n = 65), 22 and 64 unique genovars, respectively, were identified. All isolates were typeable. The present results provide evidence of a high discriminatory ability, practically the same as that for sequencing of the entire porB gene. In conclusion, the fast and high-throughput pyrosequencing technology can be used for molecular epidemiological characterization of N. gonorrhoeae.

Alleles↗
PubMed2004

Digital detection of genetic mutations using SPC-sequencing.

Deletion or insertion mutations lead to a frameshift that causes misalignment between wild-type and mutated allele sequences, making it difficult to identify such mutations unambiguously by using electrophoresis-based DNA sequencing. We have previously established the feasibility of an accurate DNA sequencing method using solid-phase capturable (SPC) dideoxynucleotides and MALDI-TOF mass spectrometry on synthetic templates, an approach we refer to as SPC-sequencing. Here, we report the application of SPC-sequencing in characterizing frameshift mutations by using the detection of the BRCA1 gene mutations 185delAG and 5382insC as examples. In this method, Sanger DNA sequencing fragments are generated in one tube by using biotinylated dideoxynucleotides. The sequencing fragments carrying a biotin moiety at the 3' end are captured on a streptavidin-coated solid phase to eliminate excess primer, primer dimers, and false stops. Only correctly terminated DNA fragments are captured, subsequently released, and analyzed by mass spectrometry to obtain digital DNA sequencing data. This method produces distinct doublet mass peaks at each point in the mass spectrum beyond the mutation site, facilitating the accurate characterization of the mutation. We have compared SPC-sequencing with electrophoresis-based sequencing in characterizing the above BRCA1 mutations, demonstrating the significant advantage offered by SPC-sequencing for the accurate identification of frameshift mutations.

DNA↗
PubMed2004

Novel mass spectrometry-based tool for genotypic identification of mycobacteria.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR amplified and in vitro-transcribed 16S rRNA gene (rDNA) was used for the identification of mycobacteria. Full-length 16S rDNA reference sequences of 12 type strains of Mycobacterium spp. frequently isolated from clinical specimens were determined by PCR, cloning, and sequencing. For MALDI-TOF MS-based comparative sequence analysis, mycobacterial 16S rDNA signature sequences ( approximately 500 bp) of the 12 type strains and 24 clinical isolates were PCR amplified using RNA promoter-tagged forward primers. T7 RNA polymerase-mediated transcription of forward strands in the presence of 5-methyl ribo-CTP maximized mass differences of fragments generated by base-specific cleavage. In vitro transcripts were subsequently treated with RNase T1, resulting in G-specific cleavage. Sample analysis by MALDI-TOF MS showed a specific mass signal pattern for each of the 12 type strains, allowing unambiguous identification. All 24 clinical isolates were identified unequivocally by comparing their detected mass signal pattern to the reference sequence-derived in silico pattern of the type strains and to the in silico mass patterns of published 16S rDNA sequences. A 16S rDNA microheterogeneity of the Mycobacterium xenopi type strain (DSM 43995) was detected by MALDI-TOF MS and later confirmed by Sanger dideoxy sequencing. In conclusion, analysis of 16S rDNA amplicons by MS after base-specific cleavage of RNA transcripts allowed fast and reliable identification of the Mycobacterium tuberculosis complex and ubiquitous mycobacteria (mycobacteria other than tuberculosis). The technology delivers an open platform for high-throughput microbial identification on the basis of any specific genotypic marker region.

Genotype↗
PubMed1995

Efficient low redundancy large-scale DNA sequencing at EMBL.

An efficient low redundancy DNA sequencing strategy should allow high accuracy determination of the consensus sequence on both strands of a DNA fragment from a minimal number of sequencing reactions with minimal overlap. At EMBL we developed a directed strategy for cosmid-scale sequencing based on primer walking, whereas most other sequencing projects of this scale rely on the random 'shotgun' strategy. In our strategy, highly accurate raw data are obtained from automated double-stranded Sanger dideoxy sequencing with inexpensive walking primers (8 to 10 $ per primer), T7 DNA polymerase and internal labelling by fluorescein-15- dATP on A.L.F. DNA sequencers (Pharmacia Biotech). The use of 60-cm long glass plates enables reading length of up to 1000 bases. Comparing various random and directed sequencing strategies in the course of the European Community yeast genome sequencing project on cosmids from chromosomes IX, XI and XV, primer walking was found to be the strategy resulting in the lowest possible redundancy of 2.6 to 2.8. Future development of the sequencing strategy is based on the new EMBL 2-dye sequencing device for simultaneous sequencing on both strands, and implementation of an initial limited random sequencing phase to reduce the number of walking primers required by a factor of 3, while still maintaining a low redundancy of approx. 3.

Animals↗
PubMed1985

[Isolation of mutant genes for human leukocyte alpha2-interferon by a method of localized mutagenesis].

An efficient method to obtain the mutant genes for human leucocyte alpha 2-interferon (IFN) has been elaborated. The technique includes the following main stages: cloning of interferon gene in M13mp8 DNA; isolation of double-stranded hybrid DNA complex, containing IFN gene as a single-stranded fragment; selective modification of a single-stranded hybrid DNA by sodium bisulphite; the repair of hybrid DNA by DNA polymerase I from Escherichia coli, transformation of Escherichia coli JN103 cells by double-stranded circular DNA, containing the selectively modified gene IFN. The technique is based on the protection of bacteriophage M13 genome from mutagen induced damage by means of converting phage DNA into the double-stranded structure leaving the single-stranded fragment to be mutagenized prone to mutagen action. This is achieved by reannealing of single-stranded M13mpB DNA hydrolyzed by restriction endonuclease BamHI. The technique preserves the infectiousness of vector DNA under the conditions permitting modification of up to 10% cytosine residues in IFN gene. Every clone resulting from transformation of Escherichia coli by modified DNA carried mutations in IFN gene, identified by sequencing after Sanger.

Base Sequence↗
PubMed1996

Construction and expression of single-chain Fv antibody against human bladder carcinoma.

We designed two sets of oligonucleotide primers to amplify the immunoglobulin heavy- and light-chain variable-region genes from genomic DNA by polymerase chain reaction (PCR). The genomic DNA was extracted from hybridoma BDI-1 cells, which secreted a monoclonal antibody (mAb) against human bladder carcinoma. The primers contained special restriction sites that allowed the variable-region genes to be easily cloned for sequencing and expression. The recombinants were sequenced by Sanger's method. It was proved that the full lengths of the VH and VK genes were 366 and 324 bp, respectively. Compared with other published sequences, the VH gene was a member of mouse heavy-chain VH subgroup II and originated from the rearrangement of VH, Dsp2.2 and JH4. The VK gene was VK subgroup IV and from VK and JK4. The VH and VK genes was inserted expression vector pWAI80. By inducement, the ScFv antibodies were expressed and secreted from Escherichia coli. Binding activities against the bladder carcinoma cells were detected. We suggest that ScFv antibody recognized the antigen specifically.

Amino Acid Sequence↗
PubMed1996

[Amplification, cloning and sequence analysis of the variable region genes of monoclonal antibody against human bladder carcinoma].

We have designed two sets of oligonucleotide primers to amplify the immunoglobulin heavy- and light-chain variable region genes from genomic DNA by the polymerase chain reaction, PCR. The genomic DNA was extracted from hybridoma BDI-1 cell, which secrets a monoclonal antibody against human bladder carcinoma. The primers contain special restriction sites that allow the variable region genes to be easy to cloning for sequencing and expression. The PCR products were ligated to plasmid PUC19. The recombinants were sequenced with Sanger's method. It was proved that a full-length VH and V kappa genes was 366 and 324 base pairs respectively. Comparaing with other published sequences, the VH gene was a member of mouse heavy-chain VH subgroup II and originated from rearrangement of VH, Dsp2.2 and JH4; the V kappa gene was V kappa subgroup IV and from V kappa and J kappa 4. It was suggested that obtained VH and V kappa genes were potentially functional genes of the monoclonal antibody against human bladder carcinoma.

Antibodies, Monoclonal↗
PubMed1997

[Gene expression of basic fibroblast growth factor receptor in bovine lens epithelial cells].

OBJECTIVE: To characterize the mechanism of promotive effects of basic fibroblast growth factor (bFGF) on proliferation of lens epithelial cells. METHODS: RNA was extracted from cultured bovine lens epithelial cells of second passage and then cDNA was synthesized with reverse transcription. cDNA segments were amplified by polymerase chain reaction using bFGF receptor oligonucleotide primer from sequence of fibroblast cells in human placenta. Then, amplified cDNA segments were cloned and sequenced by Sanger method of dideoxy-chain termination. RESULTS: It was found that the bFGF receptor mRNA in bovine lens epithelial cells was detected and partial amino-acid sequences of bovine bFGF receptor are almost the same as that of human's except 3 amino-acids. CONCLUSIONS: bFGF receptor exists in lens epithelial cells, and when bFGF is bound to its receptor, the binding plays an important role in lens epithelial cell proliferation and capsular opacity after cataract surgery.

Amino Acid Sequence↗
PubMed1990

Primer extension dideoxy chain termination nucleotide sequencing of partially purified RNA virus genomes: a technique for investigating low titre viruses with extensive genome secondary structure.

A modified version of the primer extension dideoxy chain termination nucleotide sequencing technique (Sanger et al., 1977) is described. This method has advantages over existing molecular cloning and primer extension techniques in that it allows the genome of RNA viruses to be directly sequenced from partially purified RNA preparations. Thus, viruses growing at unacceptably low titres in tissue culture can now be partially purified from infected mouse brain and sequenced. The technique also incorporates steps for the denaturation of secondary structure which has previously provided difficulties for primer extension sequencing.

Animals↗
PubMed2002

DNA sequencing with solid-phase-capturable dideoxynucleotides and energy transfer primers.

False terminations occurring in fluorescent dye-primer DNA sequencing, and nonsequencing primer extension DNA fragments generated in dye-terminator sequencing cause background noise in fluorescent electropherograms, leading to errors in sequence determination. We describe here a DNA sequencing chemistry that produces accurate and clean sequencing data on a fluorescent DNA sequencer, eliminating the false terminations and background noise. The procedure involves coupling fluorescence energy transfer (ET) primers that produce high fluorescent signals with solid-phase-capturable biotinylated dideoxynucleotides to generate Sanger DNA sequencing fragments. After the sequencing reaction,the DNA extension fragments that carry a biotin at the 3' end are captured with streptavidin-coated magnetic beads, while the other components in the sequencing reaction are washed away. Only pure DNA extension products terminated by the biotinylated dideoxynucleotides are released from the magnetic beads and are loaded onto a sequencing gel to produce accurate sequencing data.

Base Sequence↗
PubMed2001

[Gene cloning, construction and expression of single-chain Fv (scFv) against the membrane protein of Schistosoma japonicum].

OBJECTIVE: To construct single chain antibody specific to membrane protein of Schistosoma japonicum by genetic engineering technique. METHODS: The VH (heavy-chain variable region) and VL(light-chain variable region) genes were amplified by PCR from the genomic DNA of NP11-4 cell line, and sequenced by Sanger's method. The ScFv was constructed in pTHA90 vector using VH and VL genes, then expressed by IPTG. RESULTS: The VH and VL genes were obtained through PCR. The DNA sequences showed that VH and VL were new variable region genes of antibody. They were registered by GenBank. A ScFv gene with (Gly4Ser) 3 intralinker in the pTHA90 vector was successfully constructed. The ScFv was expressed as thioredoxin-fused proteins about 36.2 kDa. CONCLUSION: A specific ScFv against the membrane protein of Schistosoma japonicum was constructed and expressed.

Amino Acid Sequence↗
PubMed1977

Nucleotide sequence of the 3' terminal third of rabbit alpha-globin messenger RNA: comparison with human alpha-globin messenger RNA.

Rabbit and human alpha-globin complementary DNA was synthesized using the primer (dT10)dGdCdC hybridized to globin mRNA with reverse transcriptase, and sequenced using the plus and minus gel sequencing procedure (Sanger and Coulson, 1975; Brownlee and Cartwright, 1977). The complete 3' noncoding region, 89 nucleotides in length, and one third of the coding region of rabbit alpha-globin mRNA have thus been sequenced. These data are compared with the near complete 3' noncoding region sequence of human alpha-globin mRNA obtained in these and previous studies. The two mRNAs are approximately 80% homologous in their 3' noncoding regions, except that the human sequence has an insert of 24 nucleotides. A very similar insert in sequence has been described in human beta-globin mRNA (Proudfoot, 1977).

Animals↗
PubMed1997

[cDNA cloning, sequencing and expression of recombinant human prorelaxin].

In order to accelerate the development of biological recombinant human prorelaxin in our country, direct cloning of human prorelaxin gene from freshly prepared corpus luteum of Chinese woman by RT-PCR method was conducted. Isolated prorelaxin gene contains B, A chains and connective peptide. The amplified products were cloned and confirmed by DNA sequencing using Sanger Dideoxy method. It was subcloned into LKB2, a prokaryotic expression vector offering promoter LacI control, and highly expressed in E. coli. The results suggest that the PCR amplified DNA fragment shared identical sequence with known prorelaxin gene reported abroad. The SDS-PAGE analysis revealed that the expressed protein accounted for about 30% of the total cell protein, its MW was approximately 20 kD and conformed soluble protein.

Cloning, Molecular↗
PubMed2000

[Mutation of hepatitis B virus DNA pre-C region in patients with primary hepatocellular carcinoma in Guangxi].

OBJECTIVE: To study the association between mutation of hepatitis B virus (HBV) pre-C gene and HBV infection in patients with primary hepatocellular carcinoma (PHC) but negative hepatitis B e antigen (HBeAg) in Guangxi Province, China. METHODS: Nested polymerase chain reaction (nPCR) was used for amplification of HBV DNA Pre C region in sera collected from 16 patients with PHC in Guangxi, and then their HBV DNA nPCR products were sequenced by Sanger method. RESULTS: Sera in fourteen of 16 patients showed positive HBV DNA, with a positive proportion of 87.5% (14/16). One (C23) of them was positive for HBeAg; one (C24) was negative HBeAg, but with normal sequence in his Pre C region; two (C7, C14) were co-infected with HBV wild and mutant strains; and the remainder eight cases (C3, C4, C5, C8, C10, C11, C12, C13) were infected with mutant virus strains, but stop codon at codon 28 was only found in C4, C5 and C12. One case (C8) was co-infected with both HBV stop codon mutant and non-stop codon strains. CONCLUSION: It is uncommon for patients with PHC in Guangxi infected with HBV mutant strain with classical mutation at nt 1 896, which suggests that maybe there exist other types of mutation other than that in pre-C region causing HBV infection without HBeAg.

Base Sequence↗