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At least 649 records · Page 36Linked to original sources
PubMed1984

Nucleotide sequence of bacteriophage T4 gene 23 and the amino acid sequence of its product.

We have determined the nucleotide sequence of gene 23 of bacteriophage T4 by the methods of Maxam and Gilbert and of Sanger. The identities of approximately 80% of the amino acid residues of the major capsid protein which is encoded by gene 23 were determined additionally by Edman degradation of the intact protein and its peptides. Fifteen gene 23 amber mutation sites have been located within the sequence, and the 3' transcription termination site for genes 21, 22 and 23 has been identified.

Amino Acid Sequence↗
PubMed1987

[Molecular causes of thalassemia. IV. Cloning of the beta-globin gene in a patient with beta-thalassemia from Azerbaijan and determination of the point mutation in a minor intron].

On the basis of DNA from a beta-thalassemic patient, human gene library has been obtained using bacteriophage lambda EMBL4 as a vector. The recombinants contain human DNA insertions of 15 to 20 kb. The lambda A1 beta 1 clone has been isolated by the method of hybridization of phage plaque replicas to the HhaI fragment of JW102 plasmid containing human beta-globin cDNA. Restriction mapping revealed the presence of a 22 kb human DNA fragment comprising a portion of the beta-globin gene system. Subcloned fragments of beta-globin gene (within the pUC19 plasmid or phage MI3mp10) were sequenced using the Maxam and Gilbert method as well as that of Sanger. 2150 nucleotides in total were analysed. We have detected the point substitution G----A in the 110 nucleotide of minor intron, leading to the formation of an additional splicing site.

Bacteriophage lambda↗
PubMed1993

[Use of the polymerase chain reaction to detect beta-thalassemia mutations in heterozygous carriers from Azerbaijan while performing prenatal DNA-diagnosis].

Prenatal DNA-diagnosis of beta-thalassemia in a family from Azerbaijan revealed two mutations new for this region--G-A transition at codon 15 and G-C transversion at position 5 of the intron 1. Prenatal diagnosis was carried out by direct sequencing of in vitro amplified (PCR) beta-globin gene fragments with a modified Sanger technique using thermostable DNA polymerase. The absence of parents mutations in the fetal DNA allowed us to conclude that the fetus is normal. The diagnosis was proved at hematological testing of the baby borne.

Azerbaijan↗
PubMed1990

Characteristics of a strong promoter from figwort mosaic virus: comparison with the analogous 35S promoter from cauliflower mosaic virus and the regulated mannopine synthase promoter.

A segment of DNA from the genome of figwort mosaic virus (FMV) strain M3 possesses promoter activity when tested in electroporated protoplasts from, and transgenic plants of, Nicotiana tabacum cv. Xanthi nc. The 1.1 kb DNA segment, designated the '34S' promoter, is derived from a position on the FMV genome comparable to the position on the cauliflower mosaic virus (CaMV) genome containing the 35S promoter. The 34S and 35S promoters show approximately 63% nucleotide homology in the TATA, CCACT, and -18 to +1 domains, but in sequences further upstream the homology drops below 50%. Promoter activities were estimated using beta-glucuronidase and neomycin phosphotransferase II reporter gene systems. The activity of the 34S promoter segment approximates that of the 35S promoter in both protoplast transient expression assays and in stably transformed tobacco plants. Truncation of 5' sequences from the 34S promoter indicates that promoter strength depends upon DNA sequences located several hundred nucleotides upstream from the TATA box. In leaf tissue the 34S promoter is 20-fold more active than the mannopine synthase (MAS) promoter from Agrobacterium tumefaciens T-DNA. The 34S promoter lacks the root-specific and wound-stimulated expression of the MAS promoter, showing relatively uniform root, stem, leaf, and floral activities.

Base Sequence↗
PubMed1998

Two-dimensional electrophoresis for analysis of Mycobacterium tuberculosis culture filtrate and purification and characterization of six novel proteins.

Culture filtrate from Mycobacterium tuberculosis contains molecules which promote high levels of protective immunity in animal models of subunit vaccination against tuberculosis. We have used two-dimensional electrophoresis for analysis and purification of six novel M. tuberculosis culture filtrate proteins (CFPs): CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28. The proteins were tested for recognition by M. tuberculosis-reactive memory cells from different strains of inbred mice and for their capacity to induce a skin test response in M. tuberculosis-infected guinea pigs. CFP17, CFP20, CFP21 and CFP25 induced both a high gamma interferon release and a strong delayed-type hypersensitivity response, and CFP21 was broadly recognized by different strains of inbred mice. N-terminal sequences were obtained for the six proteins, and the corresponding genes were identified in the Sanger M. tuberculosis genome database. In parallel we established a two-dimensional electrophoresis reference map of short-term culture filtrate components and mapped novel proteins as well as already-known CFP.

Amino Acid Sequence↗
PubMed2005

A 768-lane microfabricated system for high-throughput DNA sequencing.

A 768-lane DNA sequencing system based on microfluidic plates has been designed as a near-term successor to 96-lane capillary arrays. Electrophoretic separations are implemented for the first time in large-format (25 cm x 50 cm) microdevices, with the objective of proving realistic read length, parallelism, and the scaled sample requirements for long-read de novo sequencing. Two 384-lane plates are alternatively cycled between electrophoresis and regeneration via a robotic pipettor. A total of greater than 172000 bases, 99% accuracy (corresponding to quality score 20) is achieved for each iteration of a 384 lane plate. At current operating conditions, this implies a system throughput exceeding 4 megabases of raw sequence (Phred 20) per day on the new platform. Standard operation is at "1/32x" Sanger chemistry, equal to typical genome center operation on mature capillary array machines, and a 16-fold improvement in scaling relative to previous microfabricated devices. Experiments provide evidence that sample concentration can be further reduced to 1/256x Sanger chemistry in the microdevice. Life-testing indicates a usable life of >150 hours (more than 50 runs) for the 384 lane plates. The combined advances, particularly those in read length and sample requirement, directly address the cost model requirements for adaptation of the new technology as the next step beyond capillary array instruments.

DNA↗
PubMedDate not supplied

[Protection of segments of replicative form I of phiX174 phage DNA recognized by HindII, BspRI, and AluI restrictases by Escherichia coli RNA-polymerase].

The preincubation of a DNA with E. coli RNA polymerase provides its partial protection against the HindII, BspRI and AluI cleavage giving possibility to determine the location of RNA polymerase tight-binding sites. Using this approach about 16 RNA polymerase tight-binding sites were detected on replicative form of phiX174 phage DNA. The protection degree of each of these sites depended on the preincubation conditions. Some of the protected sites hit the known phiX174 promoters and rho-dependent terminators and the other were distributed along the whole phiX174 DNA molecule. Many of them could be considered as potential promoters because they contain all the necessary elements specifying the real promoter sequences. At least some of the intrinsic promoter elements could be observed next to the rest of protected sites. One of the protected sites (R6b/l) is located in phiX174 DNA region which is very similar to the cAMP-CRP-controlled promoter sequences. It was confirmed that phiX174 DNA has two B promoters positioned by Sanger on the phiX174 nucleotide map, according to our data obtained by RNA polymerase protection experiments along with RNA product analysis of the R8 DNA fragment transcription in vitro.

Bacteriophage phi X 174↗
PubMed2000

Extensive sequence homology between the mycobacterium leprae LSR (12 kDa) antigen and its Mycobacterium tuberculosis counterpart.

The Mycobacterium leprae LSR (12 kDa) protein antigen has been reported to mimic whole cell M. leprae in T cell responses across the leprosy spectrum. In addition, B cell responses to specific sequences within the LSR antigen have been shown to be associated with immunopathological responses in leprosy patients with erythema nodosum leprosum. We have in the present study applied the M. leprae LSR DNA sequence as query to search for the presence of homologous genes within the recently completed Mycobacterium tuberculosis genome database (Sanger Centre, UK). By using the BLASTN search tool, a homologous M. tuberculosis open reading frame (336 bp), encoding a protein antigen of 12.1 kDa, was identified within the cosmid MTCY07H7B.25. The gene is designated Rv3597c within the M. tuberculosis H37Rv genome. Sequence alignment revealed 93% identity between the M. leprae and M. tuberculosis antigens at the amino acid sequence level. The finding that some B and T cell epitopes were localized to regions with amino acid substitutions may account for the putative differential responsiveness to this antigen in tuberculosis and leprosy.

Amino Acid Sequence↗
PubMed2002

A review of DNA sequencing techniques.

The four best known DNA sequencing techniques are reviewed. Important practical issues covered are read-length, speed, accuracy, throughput, cost, as well as the automation of sample handling and preparation. The methods reviewed are: (i) the Sanger method and its most important variants (enzymic methods); (ii) the Maxam & Gilbert method and other chemical methods; (iii) the Pyrosequencing method--DNA sequencing in real time by the detection of released pyrophosphate (PPi); and (iv) single molecule sequencing with exonuclease (exonuclease digestion of a single molecule composed of a single strand of fluorescently labelled deoxynucleotides). Each method is briefly described, the current literature is covered, advantages, disadvantages, and the most suitable applications of each method are discussed.

Base Sequence↗
PubMed2005

Comparative analysis of a BAC contig of porcine chromosome 13q31-q32 and human chromosome 3q21-q22.

BACKGROUND: The gene(s) encoding the ETEC F4ab/ac receptors, involved in neonatal diarrhoea in pigs (a disease not yet described in humans), is located close to the TF locus on Sscr13. In order to reveal and characterize possible candidate genes encoding these receptors, a porcine physical map of the TF region is indispensable. RESULTS: A contig of 33 BAC clones, covering approximately 1.35 Mb surrounding the TF locus on Sscr13q31-q32, was built by chromosome walking. A total of 22,552 bp from the BAC contig were sequenced and compared with database sequences to identify genes, ESTs and repeat sequences, and to anchor the contig to the syntenic region of the human genome sequence (Hsap3q21-q22). The contig was further annotated based on this human/porcine comparative map, and was also anchored to the Sanger porcine framework map and the integrated map of Sscr13 by RH mapping. CONCLUSION: The annotated contig, containing 10 genes and 2 ESTs, showed a complete conservation of linkage (gene order and orientation) with the human genome sequence, based on 46 anchor points. This underlines the importance of the human/porcine comparative map for the identification of porcine genes associated with genetic defects and economically important traits, and for assembly of the porcine genome sequence.

Animals↗
PubMed1999

Multiplexed automated DNA sequencing directly from single bacterial colonies.

Sample preparation has been one of the major bottlenecks for large-scale DNA sequencing projects in terms of time and cost. To improve sample throughput and to integrate the front-end tasks to capillary-array DNA sequencers, protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony is picked, lysis is accomplished in situ in the plastic sample tube using either a thermocycler or a heating block. Upon heating, the plasmids are released while chromosomal DNA and membrane proteins are denatured and precipitate to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed online DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. By the marriage of colony sequencing with the capillary array sequencer, both the front end and the back end of DNA sequencing are combined in a miniaturized format. This protocol will ultimately reduce the cost of sequencing to well below current levels.

DNA, Bacterial↗
PubMed1990

Partial Xq25 deletion in a family with the X-linked lymphoproliferative disease (XLP)

X-linked lymphoproliferative disease (XLP) results in exquisite vulnerability to EBV infection: fatal infectious mononucleosis (IM), acquired hypogammaglobulinemia and/or malignant lymphoma occur invariably following infection with the virus. We have identified the XLP locus using the DXS42 DNA probe having restriction length polymorphisms (RFLP). We report an interstitial deletion involving a portion of the Xq25 region in the X chromosome of an affected male, one sister, and their mother. Concordance has been established between the presence of a deletion and RFLP linkage analysis with the DXS42 probe in the kindred. This finding will contribute substantially to the mapping, cloning, and sequencing of the gene responsible for XLP.

Adult↗
PubMed2005

Entamoeba histolytica: analysis of the trophozoite proteome by two-dimensional polyacrylamide gel electrophoresis.

The Entamoeba histolytica genome project carried out at TIGR and the Sanger Institute has produced a near-complete set of deduced open reading frame sequences. These data provide strong support for the identification of signals from proteomic analyses such as two-dimensional electrophoresis by protein sequencing and/or mass spectrometric methods. To carry out an initial investigation of the E. histolytica proteome, appropriate sample preparation and silver staining protocols were adapted. After preparation of protein extracts from E. histolytica HM-1:IMSS trophozoites, solubilized proteins were separated in the first dimension in IPG (immobilized pH gradient) strips depending on their pI and subsequently in the second dimension according to their molecular weight by SDS-polyacrylamide gel electrophoresis. Of the more than 1500 protein spots visualized, several landmark spots were isolated from the gels and identified by either tryptic cleavage and subsequent MALDI-TOF mass spectrometry or by protein sequencing.

Animals↗
PubMed1999

Site-specific splice variation of the human P2X4 receptor.

P2X4 receptors are expressed in specific brain areas. We now describe site-specific splice variations of the human P2X4 receptor subunit, occurring at residue [YVIG / WVFV(W)] near the end of the first predicted transmembrane domain. p2X4(b) is formed by the insertion of an additional 16 amino acids. p2X4(C) is formed by deleting a cassette of 130 amino acids, including six of the 10 conserved extracellular cysteine residues. Transfection of P2X4(a), but not p2x4(c), formed functional channels in Xenopus oocytes and human 1321N1 cells. After transfection of p2X4(b) small, inconsistent ATP-evoked responses were detected only in the human cells, but when co-expressed, p2x4(b) may alter the function of P2X4(a) in oocytes. The distribution of splice variant RNA within human brain suggests regionally-dependent expression. These data indicate that the functions of the human P2X4 receptor may be altered by alternative splicing.

Adenosine Triphosphate↗
PubMed1997

In vitro pancreatic ductal cell carcinogenesis.

Our experiments were designed to identify initial biochemical and biological changes that occur during pancreatic carcinogenesis. TAKA-1, an immortal hamster pancreatic ductal cell line, was treated in vitro for up to 11 weeks with the pancreatic carcinogen N-nitorosobis(2-oxopropyl)amine (BOP). These treated cells were designated TAKA-1 + BOP. The growth of TAKA-1 and TAKA-1 + BOP cell lines was investigated in soft agar and in hamsters intradermally. The resulting tumor from TAKA-1 + BOP was re-cultured in vitro and designated TAKA-1 + BOP-T. Mutation of c-K-ras and p53 oncogenes, chromosomal changes, expression of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) receptor and several biochemical markers were examined in all cell lines. TAKA-1 + BOP but not TAKA-1 cells grew in soft agar and produced an invasive tumor in vivo. However, there were no differences in cell growth rate, DNA flow cytometry, or immunohistochemical findings between the non-transformed and transformed cells. TAKA-1, TAKA-1 + BOP and TAKA-1 + BOP-T cells all expressed mRNA of TGF-alpha and EGF receptor in a comparable pattern. DNA sequence analysis following polymerase chain reaction showed that neither TAKA-1 nor TAKA-1 + BOP cells has a mutation of c-K-ras or p53. Karyotype analysis demonstrated that TAKA-1 + BOP cells had more chromosomal abnormalities compared with TAKA-1 cells. Mutation of c-K-ras and p53 was not essential for carcinogenesis in hamster pancreatic ductal cells in vitro. In conclusion, immortality of the TAKA-1 cells caused expression of TGF-alpha to the same extent as in malignant cells. Chromosomal and ultrastructural patterns were the only differences detected between the non-transformed and BOP-transformed cells.

Amino Acid Sequence↗
PubMed2002

Purification and characterization of hemolymph juvenile hormone esterase from the cricket, Gryllus assimilis.

Juvenile hormone esterase (JHE) from the serum of the cricket, Gryllus assimilis, was purified to homogeneity in a four-step procedure involving polyethylene glycol precipitation, hydrophobic interaction FPLC, and ion exchange FPLC. This procedure could be completed in 4 days and resulted in a greater than 900-fold purification with greater than 30% recovery. The purified enzyme exhibited a single band on a silver-stained SDS PAGE gel and had an apparent subunit molecular mass of 52 kDa. The native subunit molecular mass, determined by gel permeation FPLC, was 98 kDa, indicating that JHE from Gryllus assimilis is a dimer of two identical or similar subunits. The turnover number of the purified enzyme (1.41 s(-1)), K(M(JH-III)) (84 +/- 12 nM) of nearly-purified enzyme, and k(cat)/K(M) (1.67 x 10(7) s(-1) M(-1)) were similar to values reported for other well-established lepidopteran and dipteran JHEs. JHE from Gryllus assimilis was strongly inhibited by the JHE transition-state analogue OTFP (octylthio-1,1,1-trifluoro-2-propanone; I(50) = 10(-7) M) and by DFP (diisopropyl fluorophosphate; I(50) = 10(-7) M). The shapes of the inhibition profiles suggest the existence of multiple binding sites for these inhibitors or multiple JHEs that differ in inhibition. Isoelectric focusing separated the purified protein into 4 isoforms with pIs ranging from 4.7-4.9. N-terminal amino acid sequences (11-20 amino acids) of the isoforms differed from each other in 1-4 positions, suggesting that the isoforms are products of the same or similar genes. Homogeneously purified JHE hydrolyzed alpha-napthyl esters, did not exhibit any detectable acetylcholinesterase, acid phosphatase, or aminopeptidase activity, and exhibited only very weak alkaline phosphatase activity. JHE exhibited a low (11 microM) K(M) for long-chain alpha-naphthyl esters, indicating that JHE may have physiological roles other than the hydrolysis of JH-III. Purification of JHE represents a key step in our attempts to identify the molecular causes of genetically-based variation in JHE activity in G. assimilis. This represents the first homogeneous purification of JHE from a hemimetabolous insect.

Acetone↗