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PubMed1988

Insertion and extension of acyclic, dideoxy, and ara nucleotides by herpesviridae, human alpha and human beta polymerases. A unique inhibition mechanism for 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate.

The ability of human alpha and beta DNA polymerases and herpes simplex virus type 2 (HSV-2) and human cytomegalovirus (HCMV) DNA polymerases to insert and extend several nucleotide analogs has been investigated using a variation of Sanger-Coulson DNA sequencing technology. The analogs included the triphosphates of two antiviral nucleosides with incomplete sugar rings: 9-(1,3-dihydroxy-2-propoxymethyl)guanine (dhpG) and 9-(2-hydroxyethoxymethyl)guanine (acyG or acyclovir), as well as dideoxy and arabinosyl nucleoside triphosphates. Three pairs of contrasting behaviors were found, each pair distinguishing the two human polymerases from the two viral ones: first, extension behavior with araNTPs; second, insertion/extension behavior with dhpGTP; and third, the relative preference for insertion of ddGTP versus acyGTP. The relative level of insertion of the nucleotide analogs by HCMV and HSV-2 DNA polymerases was dhpGTP greater than (acyGTP and araNTP) greater than ddGTP, whereas by human alpha polymerase it was araATP greater than ddGTP much greater than (acyGTP and dhpGTP) and by human beta polymerase it was (araATP and ddGTP) much greater than (acyGTP and dhpGTP). Evidence is presented for three mechanisms of inhibition by extendible nucleotides (of dhp and ara types) exhibiting frequent internalization: araATP acted as a simple pseudoterminator of alpha and beta polymerases, but was easily extended past singlet sites by Herpesviridae polymerases and only stalled at sites requiring two or more araATP insertions in a row. Herpesviridae polymerases stalled after adding dhpGMP and one additional nucleotide, suggesting that polymerase translocation problems may be a factor in polymerase inhibition by modified sugar nucleotide analogs. The amino acid sequence of the human alpha DNA polymerase, which is acyGTP resistant, was found to vary by one amino acid from the amino sequences of the Herpesviridae polymerases in a region of significant similarity and probable functional homology. Amino acid differences at that same site differentiate acyclovir-resistant HSV-1 mutants from the acyclovir-sensitive HSV-1 wild type.

Base Sequence↗
PubMed1986

[Isolation and properties of recombinant DNA from a plasmid and filamentous phage].

In the course of studying extrachromosomal DNA with composite replicons, a hybrid has been constructed by the in vitro recombination of the filamentous phage M13mp2 DNA (RF) and plasmid pUR222 (ApR). Both parental DNAs contain a fragment of lac-operon (ca. 800 bp), which includes the distal end of lacI gene, lacPO segments, and the lacZ gene proximal region coding for 145 N-terminal amino acid residues of beta-galactosidase and thus providing for alpha-complementation, the effect being cancelled with a polynucleotide insertion at the unique EcoRI site in the lacZ gene segment. E. coli BMH71-18 cells were transformed with the ligated mixture of EcoRI restricts of both DNAs. A phage-like nucleoprotein was isolated from colourless plaques (on the Xgal- and IPTG-supplemented medium); its deproteinization yielded a DNA which contains the ApR-determinant and, according to PAGE, structurally specific staining, restriction analysis, sequencing by the Sanger procedure, and electron microscopy data, is a linear double-stranded molecule comprising the phage and plasmid genomes in an equimolar ratio. Since the hybrid DNA does not display the alpha-complementation effect, both bacterial inserts are in the opposite orientation. Transformation of both phage (F+) and plasmid (F-) hosts with the hybrid DNA led to cultures which, after precipitation of the nucleoprotein from the extracellular medium and deproteinization, afforded the same composite DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Mapping↗
PubMed1986

[Variants of phage M13 DNA containing a fragment of the beta-galactosidase gene--a convenient mutation system for the study of oligonucleotide-directed mutagenesis].

A model system is developed to test oligonucleotide-directed mutations: T----C transition, T and C deletions (delta T and delta C), C insertion, double mutations (A----G, delta T), (T----C, A----G), and large oligonucleotide deletions (36 or 44 nucleotides). The system includes 9 variants of the phage M13 DNA carrying fragment of beta-galactosidase gene, and oligodeoxyribonucleotides partially noncomplementary to DNA sequence of this gene. Six variants are obtained by the site-localized mutagenesis, the other were described earlier. Induced mutations are easily tested by phenotype change of transformed bacteria (Lac+----Lac-); by formation or loss of the sites for BamHI and EcoRI restrictases; by DNA hybridization with 32P-labeled oligonucleotides; and by DNA sequencing by the Sanger method. The system is used to study the role of some factors, such as completeness of RF DNA synthesis, thermal stability of the oligonucleotide: DNA complex, quality of enzymes and substrates used in polymerase reaction, mutation type or the efficiency of mutagenesis. A number of unexpected mutations were observed in the course of oligonucleotide-directed mutagenesis. Lower yields of some mutants induced by oligonucleotides are shown to be due to the action of repair systems of bacteria.

Coliphages↗
PubMed2006

Dynamic variable selection in SNP genotype autocalling from APEX microarray data.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are DNA sequence variations, occurring when a single nucleotide--adenine (A), thymine (T), cytosine (C) or guanine (G)--is altered. Arguably, SNPs account for more than 90% of human genetic variation. Our laboratory has developed a highly redundant SNP genotyping assay consisting of multiple probes with signals from multiple channels for a single SNP, based on arrayed primer extension (APEX). This mini-sequencing method is a powerful combination of a highly parallel microarray with distinctive Sanger-based dideoxy terminator sequencing chemistry. Using this microarray platform, our current genotype calling system (known as SNP Chart) is capable of calling single SNP genotypes by manual inspection of the APEX data, which is time-consuming and exposed to user subjectivity bias. RESULTS: Using a set of 32 Coriell DNA samples plus three negative PCR controls as a training data set, we have developed a fully-automated genotyping algorithm based on simple linear discriminant analysis (LDA) using dynamic variable selection. The algorithm combines separate analyses based on the multiple probe sets to give a final posterior probability for each candidate genotype. We have tested our algorithm on a completely independent data set of 270 DNA samples, with validated genotypes, from patients admitted to the intensive care unit (ICU) of St. Paul's Hospital (plus one negative PCR control sample). Our method achieves a concordance rate of 98.9% with a 99.6% call rate for a set of 96 SNPs. By adjusting the threshold value for the final posterior probability of the called genotype, the call rate reduces to 94.9% with a higher concordance rate of 99.6%. We also reversed the two independent data sets in their training and testing roles, achieving a concordance rate up to 99.8%. CONCLUSION: The strength of this APEX chemistry-based platform is its unique redundancy having multiple probes for a single SNP. Our model-based genotype calling algorithm captures the redundancy in the system considering all the underlying probe features of a particular SNP, automatically down-weighting any 'bad data' corresponding to image artifacts on the microarray slide or failure of a specific chemistry. In this regard, our method is able to automatically select the probes which work well and reduce the effect of other so-called bad performing probes in a sample-specific manner, for any number of SNPs.

Algorithms↗
PubMed2000

Highly conserved nucleotide sequence and its deduced amino acids of the 5'-noncoding region and the capsid protein of a Bangkok isolate dengue-3 virus.

The dengue-3 virus genome encodes an uninterrupted open reading frame (ORF) flanked by 5' and 3' non-coding regions. The order of proteins encoded in dengue-3 virus ORF, as with other flaviviruses, is: Cap 5'-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3'. The nucleotide sequence of the 5'-noncoding region and the capsid protein of dengue-3virus (a Bangkok isolate: CH53489 isolated by USAMC-AFRIMS in 1973) has been analyzed in both forward and reverse directions. The PCR-based cycle sequencing technique by the enzymatic method of Sanger et al (1977) using a sequencing primer 5'-end labeled with gamma32P-ATP is the method of our choice for sequencing analysis. One cDNA template was prepared by RT-PCR technique starting from the 5'-end nucleotide 1-465 of the dengue-3 genome. In our cycle sequencing experiments, the substitution of 7-deaza-dG was used for dG in DNA eliminated much of the secondary structures that produced gel artifacts. The final sequence result of this cDNA template was established from its sequence data determined on both strands in opposite directions. Alignment between the newly established nucleotide sequence as well as its deduced amino acid sequence of the Bangkok dengue-3 virus and the published sequence data of the dengue-3 prototype (H87) was manipulated by the PC-DOS-GIBIO-DNASIS TM 06-00 (Hitachi Software). According to the deduced amino acid sequence of the Bangkok dengue-3 virus, its C protein was found to be highly positively charged because of large numbers of lysine and arginine. The homology of the nucleotide sequence between the two dengue-3 virus revealed 97%. The deduced amino acid sequences from the nucleotides 95-465 of the two viruses showed the same indicating highly conserved capsid proteins. Multiple alignment of the nucleotide sequences as well as the deduced amino acid sequences among the Bangkok dengue-3 virus and other dengue 3 viruses also confirmed the highly conserved 5'-noncoding regions and the capsid proteins.

Amino Acid Sequence↗
PubMed1980

Sequence of 1060 3'-terminal nucleotides of poliovirus RNA as determined by a modification of the dideoxynucleotide method.

The dideoxynucleotide method for sequencing DNA developed by Sanger et al. [Sanger, F., Nicklen, S. & Coulson, A. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467] was modified to allow sequence analysis of poliovirus RNA without recourse to cloning. Our method involves reverse transcription of poliovirus RNA followed by cDNA-dependent DNA synthesis in the presence of unlabeled dNTPs and 2',3'-dideoxynucleoside triphosphates, with Escherichia coli DNA polymerase I (Klenow) used to catalyze the reaction. DNA synthesis is primed by 5'-32P-labeled RNase T1- or RNase A-resistant oligonucleotides generated from poliovirus RNA. The sequence of 1060 nucleotides preceding the 3'-terminal poly(A) is presented. Based on the position of termination codons we propose that viral translation terminates at nucleotide -562.

Base Sequence↗
PubMedDate not supplied

Sequence and analysis of BECV F15 matrix protein.

Clones from the bovine enteric coronavirus (F15) cDNA library were cloned in pBR322 and sequenced by the method of Sanger and Coulson. This led to the identification of a sequence of 1,300 bases which contained a single open reading frame of 690 bases yielding a protein having properties of the matrix protein (M). It was comprised of 230 amino acids with a molecular weight of 26,376 Da. It was hydrophobic and had a net charge of +8 at neutral pH. Analysis of its secondary structure could not establish a simple transmembrane arrangement of the amino acids. Comparison of its nucleotide sequence with that of BECV Mebus strain showed only a two-base change resulting in a 100% homology between the two amino acid sequences. Furthermore, a very conserved structure of M appeared on comparison with the Dayoff optimal alignment of MHV-A59, MHV-JHM, TGEV, IBV Beaudette and IBV 6/82M amino acid sequences. As the two strains of BECV, F15 and Mebus present some antigenic differences, this led us to reconsider the role of M in viral antigen specificity. A hypothesis is that, as it seems to possess the necessary information on its transmembrane region, it is an ideal candidate for the viral budding process.

Amino Acid Sequence↗
PubMed1995

The combination of Gilbert/Maxam chemical sequencing and the dideoxynucleotide chain termination approach facilitates the construction of species specific PCR-primers based on diagnostic RAPD bands.

The randomly amplified polymorphic DNA technique (RAPD) is a modification of PCR that uses short, arbitrarily generated single primers to amplify genomic DNA. Amplified DNA-fragments are often polymorphic and can be used as individual, population- or species-specific markers. Because the RAPD technique requires a very high degree of reproducibility at the instrumentation level and with regard to buffer conditions, we propose to synthesize highly specific conventional PCR primers, the sequence of which is based on the primary diagnostic RAPD bands. In this communication we present a fast and convenient experimental strategy for converting the non-stringent RAPD conditions with their low annealing temperatures to stringent PCR conditions. Diagnostic RAPD bands were sequenced by a combination of chemical (Gilbert/Maxam) and chain termination (Sanger) techniques. Based on this sequence information, highly specific oligonucleotide primers were synthesized. The value of this approach was demonstrated for the molecular diagnosis of the important rape seed (Brassica napus) pathogen Leptosphaeria maculans.

Ascomycota↗
PubMed1998

Identification of an immunogenic histone-like protein (HLPMt) of Mycobacterium tuberculosis.

We report the identification of the first histone-like protein of Mycobacterium tuberculosis (MTB) (HLPMt). The T cell blot assay was used to identify antigens of MTB associated with human immune response in healthy contacts. Fraction 21 corresponding to proteins in the molecular weight range of approximately 30 kDa were found to be immunogenic in tuberculin reactors. None of the fractions were found to be immunogenic by this assay in non-reactors to tuberculin. All sera, irrespective of the source, showed reactivity with MTB antigen(s) over a wide molecular weight range (205-->16 kDa). In the present study fraction 21 was processed for the generation of murine polyclonal sera and amino acid sequencing. The sequence of a 16-amino acid long peptide showed a 100% homology with an open reading frame (ORF) in the translated sequence of cosmid cY349 (Sanger Centre, Cambridge, UK). The ORF was predicted to code for a protein of 214 amino acids. Oligonucleotide primers were synthesized based on the nucleotide sequence located at the 5' and 3' regions of the gene. The gene encoding the predicted protein was PCR-amplified, cloned, sequenced and expressed in Escherichia coli as a protein of 28 kDa. The expressed HLPMt protein was shown to react with the polyclonal murine sera originally raised against fraction 21. Human immune response to the recombinant HLPMt protein was demonstrated by its ability to induce lymphoproliferation in peripheral blood derived mononuclear cells, and the presence of anti-HLPMt antibodies in pooled patient sera by immunoblot. The recombinant HLPMt protein elicited a vigorous lymphoproliferative response especially in healthy tuberculin reactors compared to non-reactors and patients of tuberculosis, (P < 0.05). The protein has unique dual domains with homology to both bacterial histone-like proteins (HU) and eukaryotic histone H1. Homology to prokaryotic and eukaryotic deoxyribonucleic acid (DNA)-binding proteins suggested that HLPMt could bind DNA. DNA-binding properties were confirmed by South-Western analysis strongly suggesting an interaction between HLPMt and the MTB chromosome.

Amino Acid Sequence↗
PubMed1999

Truncated fragments in polymerase chain reaction-based DNA sequencing.

Reconstitution experiments were performed by using an ordinary dye-primer protocol of a template spiked with known amounts of truncated fragments. We observed that as little as 0.2 mole-percentage of the truncated fragment caused sequence interpretation problems. Two protocols were developed for sequencing with dye-labeled terminators; this eliminates the problems with truncated fragments, which are adapted to a one-dye chemistry. One was designed for single extension sequencing using T7 DNA polymerase and one for cycle sequencing. To avoid precipitation and centrifugation and to facilitate automation, the dye-terminator protocols included the use of a biotinylated sequencing primer. Thus, the Sanger fragments were recovered and, by magnetic separation, washed and released by formamide, EDTA, and heat treatment before loading on the electrophoresis gel. Integrated procedures for sequencing PCR products using one-dye-labeled terminators suitable for automation are described. High quality data in terms of long reads and detection of polymorphisms is obtained. The protocols serve as attractive alternatives to internal labeling and dye-primer approaches.

Base Sequence↗
PubMed1977

Nucleotide sequence (n=159) of the amino-acid-accepting 3'-OH extremity of turnip-yellow-mosaic-virus RNA and the last portion of its coat-protein cistron.

The experiments described in this paper and the following one establish the sequence of the 3'-OH terminal 159 nucleotides of turnip yellow mosaic virus RNA. Uniformly 32P-labeled turnip yellow mosaic virus RNA was partially digested with T1 ribonuclease and the fragments were fractionated by polyacrylamide gel electrophoresis. Fragments originating from the 3'-OH end of the RNA molecule were identified by testing for the 3'-terminal oligonucleotide, C-COH, after total U2 ribonuclease hydrolysis. Once identified, the 3'-OH terminal fragments were sequenced by the methods of Sanger et al. The first 51 nucleotides of the longest of the sequenced fragments (158 nucleotides) extends into the 3'-terminal part of the coat protein cistron. The coat protein cistron is followed by a stretch of 108 untranslated nucleotides whose function, though still unknown, is probably linked to the tRNA-like properties which have been attributed to the 3'-OH extremity of this viral RNA. Two possible secondary structures are proposed for the sequence and the implications of the findings with regard to the tRNA-like properties of the extremity are discussed.

Base Sequence↗
PubMed2002

[The cloning and sequencing of H-2Kk gene cDNA of 615 mice].

OBJECTIVE: The purposes of this study were to clone and sequence the major histocompatibility complex type I (MHC I) molecular antigen recognizing gene (H-2Kk) of 615 mice, and to provide the functional gene for transgenic therapy. METHODS: The 1.4 kb full-length fragment of H-2Kk gene complementary DNA (cDNA) was amplified from the total RNA of 615 mouse liver by using reverse transcription polymerase chain reaction (RT-PCR). The cDNA was inserted into PGEM3Zf(+) vector directionally, and the competent E. coli JM109 was transformed with the ligated product. The recombinant PGEM3Zf(+)-H-2Kk cDNA plasmid was obtained using restricted enzyme analysis of the transfectants. The complete sequence of 615 mouse H-2Kk cDNA was determined by using Sanger's method. RESULTS: The sequences of 615 mouse H-2Kk cDNA were 99% similar with those of H-2Kk cDNA which were reported by other researchers, and the sequences encoding antigen recognizing regions (ARS) were identical with each other. CONCLUSION: The authors cloned the MHC I molecular antigen recognizing gene (H-2Kk) of 615 mice successfully and got the functional gene of MHC I.

Animals↗
PubMed1998

Macromolecular structure and self-assembly.

The output from the molecular biology revolution has grown steadily and logarithmically from the first protein sequence, insulin (Ryle AP et al 1955 Biochem J 60:541-556), the first three-dimensional atomic structure of a macromolecule, myoglobin (Kendrew JC et al 1960 Nature 185:422-427), the first DNA gene sequence, phi X174 gene J (Sanger F et al 1977 Nature 265:687-695) and the first genome sequence for a free-living organism, Haemophilus influenzae (Fleischmann RD et al 1995 Science 269:496-512) to the current situation where the output rate is close to one new gene sequence every few minutes, several new three-dimensional structures a day and a new (bacterial) genome completed every few months. Those working in this field must readjust their horizons to this changing situation every year or two. In the area of three-dimensional structure of macromolecules and macromolecular assemblies, the methods of X-ray crystallography, nuclear magnetic resonance and electron microscopy have combined to produce powerful insights into how these molecular machines work. In this paper, I present three examples of molecular machines whose structure tells us a lot about how they work. These are the light-driven proton pump bacteriorhodopsin, the ATP synthetase molecule which contains a tiny motor and generator, and the flagellar rotary motor which provides the thrust to power physical movement of the bacterial cell. The structure itself in three-dimensional detail is thus often seen to provide the most important single insight into how things work, reducing biology to chemistry and physics. The reductionist approach in this field seems to be limited only by the accuracy by which it is possible to describe inter- and intra-molecular interactions in terms of hydrogen bonds, van der Waals interactions and electrostatic forces. At present, there is no fundamental limit in sight.

Animals↗
PubMed2001

[A mutation detection in a transcription factor for adipocyte development in children with severe obesity.].

OBJECTIVE: A substantial proportion of human obesity may be explained by genetic variability. Researchers have tried to identify the important genes in obesity with little sucsess. PPARg2 (peroxisome proliferator activated receptor g 2) is a transcription factor of the nuclear hormone receptor superfamily. It plays a key role in the developement and differentiation of adipocytes. Recently the mutation Pro115Gln in the PPARg2 gene was identified and shown to have a significant correlation with severe obesity. The actual prevalence and distribution of this mutation is not known. The aim of this study was to look for this mutation among Icelandic children suffering from severe obesity. MATERIAL AND METHODS: Thirty-five children and adolescents, aged 4-18, who have been diagnosed with severe obesity participated in the study. Eight parents and siblings aged 19-41 also participated. All study subjects had been obese since early childhood. Body mass index (BMI) was used to describe the phenotype of the subjects. The participants had a BMI of 28.0 to 52.2 kg/m(2). Genomic DNA was extracted from leucocytes. A 131 bp segment was amplified using polymerase chain reaction. The amplified product was digested with the restriction enzyme Hinc II, resolved on agarose gel and visualized under ultraviolet illumination after staining with ethidium bromide. To examine other mutations on the same 131 bp segment enzymatic mutation detection (EMD) was used. Finally the segments giving variable results using EMD were sequenced using the classic Sanger s method. RESULTS: The mutation Pro115Gln was not found in any of the specimens after analysis of the restriction fragment length polymorphism. The results of EMD indicated mutations or polymorphisms in three of the subjects but DNA sequencing failed to confirm these results. CONCLUSIONS: The mutation Pro115Gln or other genetic alternations within the exon examined do not appear to have a significant role in severe early - onset obesity in Icelandic children.

English Abstract↗
PubMed2006

[Construction and identification of eukaryotic expression plasmid pcDNA3. 1-BACE and its transient expression in COS-7 cells].

OBJECTIVE: To generate eukaryotic expression vector of pcDNA3. 1-beta-site amyloid precursor protein cleaving enzyme (BACE) and obtain its transient expression in COS-7 cells. METHODS: A 1.5 kb cDNA fragment was amplified from the total RNA of the human neuroblastoma cells by the RT-PCR method and was cloned into the plasmid pcDNA3.1. The vector was identified by the double digestion with restriction enzymes BamHI and XhoI and was sequenced by the Sanger-dideoxy-mediated chain termination. The expression of the BACE gene was detected by immunocytochemistry. RESULTS: The results showed that the cDNA fragment included 1.5 kb total coding region. The recombinant eukaryotic cell expression vector of pcDNA3. 1-BACE was constructed successfully, and the sequence of insert was identical to the published sequence. The COS-7 cells transfected with the pcDNA3. 1-BACE plasmid expressed a high level of the BACE protein in the cytoplasm. CONCLUSION: The recombinant plasmid pcDNA3. 1-BACE can provide a very useful tool for the research on the cause of Alzheimer's disease and lay an important foundation for preventing Alzheimer's disease.

Amyloid Precursor Protein Secretases↗
PubMed1987

Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants.

Messenger RNAs encoding the nitrate reductase apoenzyme from tobacco can be translated in a cell-free system. Poly(A)+ mRNA fractions from the 23-32 S area of a sucrose gradient were used to build a cDNA library in the expression vector gt11 with an efficiency of cloning of approximately 10(4) recombinants/ng mRNA. Recombinant clones were screened with a rabbit polyclonal antibody directed against the corn nitrate reductase, which cross reacts specifically with the nitrate reductases from dicotyledons. Among 240000 recombinant plaques, eight clones were isolated containing inserts of sizes ranging from 1.6 kb to 2.1 kb and sharing sequence homologies. Seven of these clones contained a common internal 1.6 kb EcoRI fragment. The identity of these clones was confirmed as follows. A fusion protein of 170 kDa inducible by IPTG and recognized by the rabbit nitrate reductase antibody was expressed by a lysogen derived from one of the recombinants. The antibodies binding the fused protein were eluted and shown to be inhibitory to the catalytic activity of tobacco nitrate reductase. Two monoclonal antibodies directed against nitrate reductase were also able to bind the hybrid protein. The 1.6 kb EcoRI fragment was sequenced by the method of Sanger. The open reading frame corresponding to a translational fusion with the -galactosidase coding sequence of the vector shared strong homology at the amino acid level with the heme-binding domain of proteins of the cytochrome b5 superfamily and with human erythrocyte cytochrome b5 reductase. When the 1.6 kb EcoRI fragment was used as a probe for Northern blot experiments a signal corresponding to a 3.5 kb RNA was detected in tobacco and in Nicotiana plumbaginifolia mRNA preparations but no cross-hybridization with corn mRNAs was detected. The probe hybridized with low copy number sequences in genomic blots of tobacco DNA.

Amino Acid Sequence↗