Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Dideoxynucleotides”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9Linked to original sources
PubMed1984

Sequence comparison of the 3' ends of a subgenomic RNA and the genomic RNAs of barley stripe mosaic virus.

All strains of barley stripe mosaic virus examined encapsidate small amounts of an 800-nucleotide (NT) gamma-subgenomic (sg) RNA. This sgRNA has been isolated from genomic (g) RNAs of the Type and North Dakota 18 (ND18) strains and the sequence of these RNAs has been compared near the 3' end. The immediate 3' termini of the gRNAs terminate in the icosomer-GGUCCCCCAAGGGAAGACCAOH-3' and differ from the sgRNAs, which are polyadenylated. The poly(A) tracts of the sgRNAs are heterogeneous with lengths ranging from 10 to greater than 150 NT. Polyacrylamide gel electrophoresis of complementary (c) DNAs transcribed in the presence of dideoxynucleotides reveals that the sgRNAs from Type and ND18 have almost identical sequences for at least 160 NT adjacent to the 5' side of the poly(A) region. This region of the sgRNA from the ND18 strain is nearly identical to a 95-NT sequence adjacent to a poly(A) tract located at the 3' end of a 2050-base pair cDNA cloned from the gamma-genomic RNA of ND18. These results suggest that the sequences encoding the sgRNA are located upstream of an internal poly(A) region situated more than 200 NT from the 3' end of the gamma-genomic RNA.

Base Sequence↗
PubMed1985

Polytranscripts of Sendai virus do not contain intervening polyadenylate sequences.

Discrete high-molecular-weight RNA species with the properties of polytranscripts were observed in poly(A)-rich RNA extracted from Sendai virus-infected cells. These RNA species were virus specific, being synthesized in the presence of actinomycin D, but not seen in uninfected cells. They were not genome or antigenome fragments, since they were not encapsidated, as shown by their destruction when ribonuclease was added to cell homogenates and by their absence from the RNA fractions that did not bind to oligo(dT)-cellulose. Two lines of evidence indicated that the gene-specific regions of these polytranscripts were not linked by poly(A) sequences, but were faithful copies of virus genomic RNA sequences at gene boundaries. First, a small cDNA clone obtained by reverse transcription of poly(A)-rich RNA species from infected cells contained 90 bases from the 5' terminus of the gene for the P protein and about 600 bases from the 3' end of the downstream gene, which specifies the M protein, the entire cloned sequence being an accurate complement of the genomic RNA. Second, dideoxynucleotide sequencing of poly(A)-rich RNA species primed by virus gene-specific oligodeoxynucleotides revealed read-through products of transcription containing no detectable poly(A). If Sendai virus polytranscripts are intermediates in the production of monocistronic viral mRNAs by a cleavage process, and poly(A) sequences do not link the mRNAs, polyadenylation would have to follow the cleavage step; it seems more likely that these polytranscipts are aberrant transcription products generated by occasional termination failure in a stop-start mechanism of transcription.

Base Sequence↗
PubMed1985

Nucleotide sequences of the 1B and 1C nonstructural protein mRNAs of human respiratory syncytial virus.

The genes encoding the 1C and 1B mRNAs of human respiratory syncytial (RS) virus are first in the order of viral transcription and encode nonstructural (NS) proteins of approximate molecular weights 14,000 and 11,000, respectively, estimated by gel electrophoresis. The complete nucleotide sequences of the 1C and 1B mRNAs determined from several full-length cDNA clones are described. The 1C and 1B mRNAs contain 528 and 499 nucleotides, respectively, exclusive of poly(A), and encode proteins of 139 and 124 amino acids. The calculated molecular weights of the predicted NS1C and NS1B proteins are 15,567 and 14,674, respectively. Both mRNA sequences contain the 5'-terminal sequence, 5' GGGGCAAAU . . . , and the 3'-terminal sequence, 5' . . . AGUAUA(N)1-4-poly(A), that were identified previously as conserved among six other RS viral mRNAs. In addition, a dicistronic readthrough RNA having the general structure 5' 1C mRNA-intergenic sequence-1B mRNA 3' was identified by dideoxynucleotide sequencing of intracellular poly(A)+ RNA using a DNA primer derived from a 1B-cDNA clone. In the dicistronic RNA, the nucleotide sequences of the 1C and 1B cistrons are separated by, in mRNA sense, four A residues and the intergenic sequence 5' . . . CUUAACAGAAGACAAAAAN . . . 3' (N represents unidentified nucleotide). The significance of these sequences is discussed.

Base Sequence↗
PubMed1986

Fusion glycoprotein of human parainfluenza virus type 3: nucleotide sequence of the gene, direct identification of the cleavage-activation site, and comparison with other paramyxoviruses.

The complete sequences of the fusion (F) mRNA and protein of human parainfluenza virus type 3 (PF3) were determined from overlapping cDNA clones. To confirm the cDNA sequence, the complete sequence of the F gene was determined independently by dideoxynucleotide sequencing of genomic RNA using synthetic oligonucleotide primers. The mRNA contains 1845 nucleotides, exclusive of poly (A), has an unusually long (193-nucleotide) 5' nontranslated region, and encodes an F0 protein of 539 amino acids. The site within F0 of the proteolytic cleavage that activates fusion activity was established by direct amino acid sequencing of the NH2 terminus of the F1 subunit. The PF3 F0 protein shares major structural features with the previously sequenced F0 proteins of Sendai virus (murine parainfluenza type 1) and simian virus 5 (SV5, canine parainfluenza type 2), including: similarity in overall length; similarity in location of the site of the activating proteolytic cleavage; the presence of an NH2-terminal signal peptide and COOH-proximal membrane anchor; strong conservation of the sequence at the NH2 terminus of the F1 subunit; and nearly exact conservation in the number and positions of cysteine residues. Alignment of the F0 protein sequences of PF3 with those of Sendai, SV5, and respiratory syncytial virus (RSV) using a matrix that scores both amino acid matches and mismatches provided highly significant statistical evidence that all four proteins are related. The order of decreasing relatedness to PF3 was found to be: Sendai virus, SV5, and RSV.

Amino Acid Sequence↗
PubMed1987

Complete nucleotide sequence of the tick-borne, orthomyxo-like Dhori/Indian/1313/61 virus nucleoprotein gene.

The complete nucleotide sequence of the fifth largest segment of single-stranded RNA of the tick-borne, orthomyxo-like Dhori/Ind/1313/61 virus was determined by using cloned cDNA derived from infected cell mRNA and dideoxynucleotide sequencing of viral RNA. The fifth RNA contains 1479 nucleotides and can code for a protein of 477 amino acids with a molecular weight of 53,679 Da. The RNA 5 protein of the Dhori/Ind/1313/61 virus possesses five short regions (16-26 amino acids) which share a high degree (50-59%) of amino acid sequence homology with a computer-aligned consensus sequence of the influenza nucleoprotein gene family. These and other structural features of the RNA 5 protein suggest that RNA 5 of Dhori viruses codes for the nucleoprotein. The data also suggest that Dhori viruses are orthomyxoviruses, but that they are more distantly related to the influenza viruses than type A, B, and C viruses are to each other.

Base Sequence↗
PubMed1989

The genome structure of turnip crinkle virus.

The nucleotide sequence of turnip crinkle virus (TCV) genomic RNA has been determined from cDNA clones representing most of the genome. Segments were confirmed using dideoxynucleotide sequencing directly from viral RNA, and the 3' terminal sequence was confirmed by chemical sequencing of end-labeled genomic RNA. Three open reading frames (ORFs) have been identified by examination of the deduced amino acid sequences and by comparison with the ORFs found in the genome of carnation mottle virus. ORF 1 initiates near the 5' terminus of the genome and is punctuated by an amber termination codon. Translation of ORF 1 would yield a 28-kDa protein and an 88-kDa read-through product. The read-through domain possesses amino acid sequence similarities with putative viral RNA polymerases. ORFs 2 and 3 encode products of 38 (coat protein) and 8 kDa, respectively, which are expressed from subgenomic mRNAs. The organization of the TCV genome suggests that TCV is closely related to carnation mottle virus and distinct from members classified in other small RNA virus groups, such as the tombus- and sobemoviruses.

Amino Acid Sequence↗
PubMed1989

Heterogeneity in mRNA of human papillomavirus type-6 subtypes in respiratory tract lesions.

We have analyzed the structure of viral transcripts in six HPV-6c-induced respiratory tract lesions, which included four benign laryngeal papilloma, one benign nasopapilloma, and one malignant tumor, in four benign laryngeal papilloma induced by HPV-6e, and in one benign laryngeal papilloma induced by HPV-6f. Northern analysis and S1 nuclease digestion with subgenomic RNA probes demonstrated that the major exon of 1050 bases had a 5' end in the E4 open reading frame and a 3' end in E5B. Primer extension from a synthetic oligonucleotide in E4 was used to examine sequences 5' to the major exon. Differences were found in length, start sites, and relative abundance of the first exon in transcripts produced in HPV-6c-induced infections as compared to HPV-6e- and HPV-6f-induced infections. Primer extension in the presence of dideoxynucleotides facilitated sequence analyses of the first exon. HPV-6c transcripts contained common sequences from E1 that in different transcripts extended farther upstream in the 5' direction into E7 resulting in different lengths. All of the mRNAs had the same splice junction at nucleotide 847 in E1 and 3325 in E4. Similarly, the HPV-6e transcripts shared common sequences in the first exon that differed in length as a result of different starting points and had the same splice junction as the HPV-6c transcripts. No differences were found in the structure of viral transcripts in a malignant vs benign lesions, nor in those of nasopapilloma vs laryngeal papilloma when induced by the same HPV-6 subtype.

Autoradiography↗
PubMed1990

Molecular cloning and sequence analysis of the fusion glycoprotein gene of human parainfluenza virus type 2.

A cDNA clone containing a 2.0-kb insert was identified as the human parainfluenza virus type 2 (PI2) fusion glycoprotein gene by hybridizing with a viral RNA probe and a synthetic oligonucleotide derived from a conserved sequence found in other paramyxovirus fusion protein genes. The complete nucleotide sequence of the glycoprotein gene was determined by the dideoxynucleotide sequencing procedure and found to contain a single, large open reading frame encoding a protein of 551 amino acids with a calculated molecular weight of 59,664. Comparison of the P12 fusion protein with those of other paramyxoviruses indicated similarities in overall length, N-terminal signal peptide sequence (amino acids 7 to 25), C-terminal membrane-spanning region (amino acids 486 to 513), and a highly conserved fusion sequence region at the N-terminus of the F1 subunit (amino acids 107 to 132).

Amino Acid Sequence↗
PubMed1991

Nucleotide sequences of the 3' leader and 5' trailer regions of human respiratory syncytial virus genomic RNA.

The nucleotide sequences of the 3' extracistronic (leader) and 5' extracistronic (trailer) regions were determined for genomic RNA (vRNA) of human respiratory syncytial virus (RSV) strain A2. To sequence the 3' leader region, vRNA was extracted from purified virions, size-selected, polyadenylated, copied into cDNA, amplified by the polymerase chain reaction, cloned, and sequenced. The 3' leader sequence is 44 nt, which is somewhat shorter than its counterparts (50 to 70 nt) in other nonsegmented negative-strand viruses sequenced to date. The 5' trailer region was mapped and sequenced in part directly by dideoxynucleotide sequencing of vRNA. The sequence was confirmed and completed by analysis of cDNA clones derived from vRNA. The 5' trailer sequence is 155 nt in length, which is substantially longer than its counterparts (40 to 70 nt) in other nonsegmented negative-strand viruses. Ten of the 11 terminal nt of the 3' leader and 5' trailer regions were complementary. Among the other paramyxoviruses, the terminal 5 to 16 nt of the leader and trailer regions are highly conserved, but the corresponding RSV sequences were identical to the others only for the terminal 2 nt of each end. Surprisingly, the termini of the RSV leader and trailer regions were in somewhat better agreement with those of the rhabdoviruses vesicular stomatitis virus and rabies virus, sharing identity for the first 3 or 4 nt.

Base Sequence↗
PubMed1992

Structure of the L (polymerase) protein gene of sonchus yellow net virus.

The complete nucleotide sequence of the L protein gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was determined by dideoxynucleotide sequencing of cloned cDNAs derived from the negative-strand genomic RNA. The L protein gene is composed of 6401 nucleotides (nt) located between positions 7158 and 13558 relative to the 3' end of the genomic RNA. Sequence analysis suggests that the complementary mRNA contains a 44 nt untranslated 5' leader sequence preceding an open reading frame of 6348 nucleotides that is capable of encoding a polypeptide of 2116 amino acids with a deduced molecular weight of 241,569 Da. The L protein is positively charged, has a high proportion of the amino acids Leu and Ile, and contains putative polymerase and RNA binding domains. Extended alignment of the SYNV L protein amino acid sequence with those of other nonsegmented negative-strand RNA virus polymerases reveals conservation of sequences within 12 blocks that appear sequentially along the protein. A cluster dendrogram derived from the L protein alignments indicates that SYNV is more closely related to animal rhabdoviruses than to the paramyxoviruses and that the animal rhabdoviruses have diverged less from each other than from SYNV.

Amino Acid Sequence↗
PubMed1980

Mutations at the yeast SUP4 tRNATyr locus: DNA sequence changes in mutants lacking suppressor activity.

Yeast strains harboring indepjendent mutations within the SUP4 tyrosine tRNA gene have been selected by virtue of their inactivating effect upon the SUP4-o UAA suppressor. Three fourths of the mutations at SUP4 are point alterations; the rest resemble the deletions described by Rothstein (1979). A meiotic genetic fine structure map of the locus was made by crossing 69 of the mutants in all combinations and testing for the frequency of SUP4-o recombinants. The sequences of SUP4 genes cloned from 32 mutant strains were determined by the dideoxynucleotide terminator method, using as primer a synthetic oligodeoxynucleotide corresponding to a sequence adjoining the SUP4 3' terminus. The positions of the DNA sequence alterations showed good colinearity with the positions of the mutations on the genetic map. One of the 26 mutant sites found by DNA sequencing lies within the intervening sequence. At this site three repeat mutations were found, each changing AT leads to TA. Whereas mutations were generally rather uniformly distributed throughout the tRNATyr coding sequence, none occurred in the DNA sequences flanking the mature tRNATyr sequence or in a 12 nucleotide sequence including the 10 bp which constitute the 3' side of the intervening sequence.

Base Sequence↗
PubMed1987

Development of a yeast system to assay mutational specificity.

We have developed a system wherein DNA alterations occurring in a target gene in the yeast Saccharomyces cerevisiae can be determined by DNA sequencing. The target gene, SUP4-o, an ochre suppressor allele of a yeast tyrosine tRNA gene, has been inserted into a shuttle vector (YCpMP2) which is maintained in yeast at a copy number of one per cell Mutations in SUP4-o are selected by virtue of their inactivation of suppressor activity. Rapid DNA preparations from these mutants are used to transform an appropriate bacterial strain. Since YCpMP2 also carries the M13 phage replication origin, superinfection of bacterial cells containing the plasmid with wild-type M13 phage yields single stranded YCpMP2 DNA suitable for dideoxynucleotide chain termination sequencing. We have used this system to examine mutations arising spontaneously in the SUP4-o gene. The spontaneous mutants occurred at a frequency of 3.2 X 10(-6)/viable cell, corresponding to a rate of 2.7 X 10(-7) events/cell division. Following bacterial transformation, 16% of the recovered plasmids tested displayed altered gel mobility consistent with loss of significant portions of the plasmid. Hybridization analysis of total yeast DNA and use of purified YCpMP2 revealed that these very large deletions were not generated in yeast but were associated with bacterial transformation. Among the SUP4-o mutants analyzed by DNA sequencing, we identified each type of single base pair substitution (transitions and transversions), small deletions of varying length (1-32 base pairs) and more extensive deletions of undetermined size. These results demonstrate that the SUP4-o system can be used to detect various types of mutation at numerous sites in a single eukaryotic gene and to characterize the DNA sequence changes responsible for the mutations selected.

DNA Damage↗
PubMed1992

Rapid sequencing of the Sendai virus 6.8 kb large (L) gene through primer walking with an automated DNA sequencer.

The determination of the complete DNA sequence of the large (L) polymerase gene of Sendai virus strain Fushimi was used to explore the potential and feasibility of primer walking with fluorescent dye-labelled dideoxynucleotide terminators on an automated ABI DNA sequencer. The rapid identification of the complete sequence demonstrated that this approach is a time- and cost-saving alternative to classical sequencing techniques. Analysis of the data revealed that the L gene of Sendai virus strain Fushimi consists of exactly 6800 nucleotides and that the deduced amino acid sequence identifies a single open reading frame encoding a protein of 252.876 kDa. In contrast to Sendai virus strain Enders, the L mRNA of strain Fushimi is monocistronic. The comparison of the deduced amino acid sequences of the L genes of three different Sendai virus strains confirmed the existence of conserved as well as variable regions in the L protein and revealed a high grade of conservation in the carboxyterminal third. Furthermore, functional amino acid sequence motifs, like elements of RNA-dependent RNA polymerases and ATP-binding sites as postulated previously, were identified.

Base Sequence↗
PubMed1986

Phylogeny of helminths determined by rRNA sequence comparison.

The nucleotide sequence of the 5' ends of the 28S-like rRNA molecules of five species of helminths was determined directly, using a variation on the dideoxynucleotide chain-termination method which requires only 10 micrograms of total cellular RNA for analysis. Nucleotide sequence comparisons over 208 bases allowed the phylogeny of these organisms to be determined. The data show that the rRNA sequence of Nematospiroides dubius, a nematode, is as divergent from that of two platyhelminths, Hymenolepis diminuta and Schistosoma mansoni, as it is from the rRNA sequence of the two nematodes Onchocerca gibsoni and Brugia pahangi. The latter two appear to be very closely related, whereas the two platyhelminths are more distant from each other. The study demonstrates the usefulness and generality of rRNA sequencing for the systematic phylogenetic classification of parasitic organisms whose tissues are only available in relatively small amounts.

Animals↗
PubMed1991

Purification and characterization of DNA polymerases from Plasmodium berghei.

DNA polymerases from the malaria parasite Plasmodium berghei were purified more than 50-fold. Several distinct enzymatic activities were isolated that could be distinguished by the use of various specific DNA polymerase inhibitors. In particular, subdivision into an aphidicolin-sensitive and an aphidicolin-resistant group was possible. Further analysis allowed a better comparison with host DNA polymerases and indicated that one aphidicolin-sensitive DNA polymerase resembled DNA polymerase alpha displaying processive DNA synthesis and using RNA primers, whereas another aphidicolin-sensitive DNA polymerase was distributive and only used DNA primers. Marked differences from the host enzymes do exist, however, such as insensitivity to BuPdGTP. Another P. berghei DNA polymerase was isolated that showed characteristics of a DNA polymerase beta-like enzyme, but which differed from host DNA polymerase beta in its insensitivity to dideoxynucleotides.

Animals↗
PubMed1983

Nucleotide sequence of the herpes simplex virus type 2 (HSV-2) thymidine kinase gene and predicted amino acid sequence of thymidine kinase polypeptide and its comparison with the HSV-1 thymidine kinase gene.

To analyze the boundaries of the functional coding region of the HSV-2(333) thymidine kinase gene (TK gene), deletion mutants of hybrid plasmid pMAR401 H2G, which contains the 17.5 kbp BglII-G fragment of HSV-2 DNA, were prepared and tested for capacity to transform LM(TK-) cells to the thymidine kinase-positive phenotype. These studies showed that hybrid plasmids containing 2.2-2.4 kbp subfragments of HSV-2 BglII-G DNA transformed LM(TK-) cells to the thymidine kinase-positive phenotype and suggested that the region critical for transformation might be less than 2 kbp. That the activity expressed in the transformants was HSV-2 thymidine kinase was shown by experiments with type-specific enzyme-inhibiting rabbit antisera and by disc-polyacrylamide gel electrophoresis analyses. DNA fragments of the HSV-2 TK gene were subcloned in phage M13mp9 and M13mp8. A sequence of 1656 bp containing the entire coding region of the TK gene and the flanking sequences was determined by the dideoxynucleotide chain termination method. Comparisons with the HSV-1(Cl 101) TK gene revealed that PstI, PvuII, and EcoRI cleavage sites had homologous locations as did promoter, translational start and stop, and polyadenylation signals. Extensive homology was observed in the nucleotide sequence preceding the ATG translational start signal and in portions of the coding region of the genes. Comparisons of the predicted amino acid sequences of the HSV-1 and HSV-2 thymidine kinase polypeptides revealed that both were enriched in alanine, arginine, glycine, leucine, and proline residues and that clear, but interrupted homology existed within several regions of the polypeptide chains. Stretches of 15-30 amino acid residues were identical in conserved regions. The possibility is suggested that domains containing some of the conserved amino acid sequences might have a role in substrate binding and as major antigenic determinants.

Amino Acid Sequence↗
PubMed1992

Sequencing of the primary adhesion domain of bovine von Willebrand factor.

A cDNA library, constructed from bovine heart endothelial cell poly(A)+ RNA, was screened using a BstXI fragment of human von Willebrand and factor (vWF) cDNA as a probe. This probe codes for the major adhesion domain of vWF that includes the GPIb, collagen and heparin binding domains. Of the ten positive clones obtained, a clone that spanned the region of interest was sequenced by the dideoxynucleotide method yielding a sequence of 1550 bp. This region of the bovine cDNA codes for amino acids corresponding to #262 to #777 in human vWF and encompasses the entire pro adhesion domain. Both the nucleotide sequence and the deduced amino acid sequence are 82% homologous to those of human vWF. Cysteine residues #471, 474, 509 and 695, which form intrachain bonds in human vWF, are also present in the bovine vWF sequence.

Amino Acid Sequence↗
PubMed1992

Complete cDNA sequence of bovine alpha 1-antitrypsin.

A cDNA clone coding for the entire bovine alpha 1-antitrypsin molecule has been isolated from a lambda gt11 bovine liver cDNA library using a human alpha 1-antitrypsin cDNA as a probe. The bovine cDNA was sequenced by the dideoxynucleotide chain termination method. Comparison of the translated amino acid sequence of the bovine alpha 1-antitrypsin with those of the human, baboon, sheep, rat and mouse demonstrates the preservation of most of the critical structural determinants. The bovine and the sheep molecules have a sequence homology of 94% and both the molecules contain four cysteine residues; there is only one cysteine in the others.

Amino Acid Sequence↗