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Biomedical subjects

B Moss

Publications and source records attributed to B Moss.

At least 397 records · Page 22Linked to original sources
PubMed1984

Regulation of expression and nucleotide sequence of a late vaccinia virus gene.

A subset of vaccinia virus genes are expressed only after DNA replication. To investigate the regulation of such transcriptional units, a representative gene encoding a major late polypeptide (Mr, 28,000) was mapped and sequenced. Translatable mRNAs were heterogeneous in length and overlapped several early genes downstream. The 5' end of the message was located, and the DNA segment upstream was excised and ligated to the coding sequence of the easily assayable procaryotic chloramphenicol acetyltransferase gene. The resulting chimeric gene was recombined into the thymidine kinase locus of the vaccinia virus genome, and infectious recombinant virus was isolated. Both the time of chloramphenicol acetyltransferase synthesis in infected cells and the requirement for DNA replication indicate that the sequence upstream of the late gene contains cis-acting transcriptional regulatory signals.

Base Sequence↗
PubMed1983

Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen.

Potential live vaccines against hepatitis B virus have been produced. The coding sequence for hepatitis B virus surface antigen (HBsAg) has been inserted into the vaccinia virus genome under control of vaccinia virus early promoters. Cells infected with these vaccinia virus recombinants synthesize and excrete HBsAg and vaccinated rabbits rapidly produce antibodies to HBsAg.

Animals↗
PubMed1983

Selective transcription of vaccinia virus genes in template dependent soluble extracts of infected cells.

A soluble system that specifically and accurately initiates transcription on defined vaccinia virus templates has been obtained from lysates of infected cells. The required regulatory signals are contained within a DNA segment extending about 230 bp upstream and 30 bp downstream of the RNA start site. Transcription is resistant to alpha-amanitin and inhibited by antibodies to the viral RNA polymerase. Whole cell extracts from uninfected cells cannot accurately transcribe vaccinia DNA. Conversely, extracts prepared at 2 hr or later after vaccinia infection no longer transcribe RNA polymerase II templates but retain the ability to transcribe RNA polymerase III templates as well as vaccinia virus DNA. These profound changes in transcriptional specificity may contribute to the selective expression of viral genes following vaccinia infection.

Base Sequence↗
PubMed1983

Infectious poxvirus vectors have capacity for at least 25 000 base pairs of foreign DNA.

To test the capacity of poxviruses for added foreign DNA, a recombinant was constructed that contains 24 700 bp of bacteriophage lambda DNA inserted within the vaccinia virus thymidine kinase (TK) gene. The recombinant is stable, infectious and replicates in tissue culture at the same rate and to the same titer as standard vaccinia virus. This size flexibility of the poxvirus genome and the lack of stringent packaging requirements are useful features for an infectious eukaryotic cloning vector.

Bacteriophage lambda↗
PubMed1983

Construction and characterization of an infectious vaccinia virus recombinant that expresses the influenza hemagglutinin gene and induces resistance to influenza virus infection in hamsters.

A DNA copy of the influenza virus hemagglutinin gene, derived from influenza virus A/Jap/305/57 (H2N2) was inserted into the genome of vaccinia virus under the control of an early vaccinia virus promoter. Tissue culture cells infected with the purified recombinant virus synthesized influenza hemagglutinin, which was glycosylated and transported to the cell surface where it could be cleaved with trypsin into HA1 and HA2 subunits. Rabbits and hamsters inoculated intradermally with recombinant virus produced circulating antibodies that inhibited hemagglutination by influenza virus. Furthermore, vaccinated hamsters achieved levels of antibody similar to those obtained upon primary infection with influenza virus and were protected against respiratory infection with the A/Jap/305/57 influenza virus.

Animals↗
PubMed1983

Vaccinia virus thymidine kinase and neighboring genes: mRNAs and polypeptides of wild-type virus and putative nonsense mutants.

Enzymatically active thymidine kinase (TK) was made in reticulocyte lysates programmed with early vaccinia mRNA that hybridized to plasmid recombinants containing either of two adjacent small DNA subsegments of the viral HindIII-J fragment. The map position of an early polypeptide, with a molecular weight of 19,000 (19K), coincided precisely with that of the TK. The absence of the 19K polypeptide in cell-free translation products of hybridization-selected mRNAs from several TK-negative mutants provided an independent identification of the TK polypeptide. The small size of the TK polypeptide of vaccinia virus distinguishes it from that of procaryotes, eucaryotes, and herpesvirus. Five early mRNAs of 3,840, 2,390, 1,790, 1,070, and 590 nucleotides were mapped within the HindIII-J fragment by RNA blotting and nuclease S1 digestion of RNA-DNA hybrids. The RNAs of 590 and 2,380 nucleotides were found to have 5' coterminal ends and represent major and minor forms, respectively, of the TK message. The 3' end of the minor TK mRNA appeared to be coterminal with the 3' end of the 1,790-nucleotide transcript which encodes a 41K polypeptide. The 1,070-nucleotide RNA was identified as the message for a 21K polypeptide. All of these RNAs, including the two forms of the TK message, were made by the putative TK-negative nonsense mutants.

DNA Restriction Enzymes↗
PubMed1983

Nucleotide sequence of the vaccinia virus thymidine kinase gene and the nature of spontaneous frameshift mutations.

Nucleotide sequencing of a 1,300-base-pair vaccinia virus DNA segment previously shown to contain a thymidine kinase (TK) gene revealed an uninterrupted reading frame of 177 codons capable of producing a polypeptide with a molecular weight of 20,102. Mapping of the TK mRNA by primer extension indicated a unique 5' end that precedes the initiation codon by only six nucleotides. Multiple 3' ends within a 10-nucleotide region, about 30 nucleotides beyond the termination codon, were located by nuclease digestion of DNA-RNA hybrids, and the length of the TK transcript, exclusive of polyadenylate, was estimated to be approximately 570 nucleotides. The region preceding the TK mRNA start site is extremely A + T rich and has sequence homologies with three other early genes. Genetic information is so compressed in this region of the DNA that the putative transcriptional regulatory sequence of the TK gene overlaps the coding sequence of a late gene. Only nine nucleotides separate the termination codon of the late gene from the initiation codon of the TK gene. Downstream, 66 nucleotides separate the TK termination codon from the apparent initiation codon of another early gene. The nature of three independent TK- mutants was revealed by nucleotide sequencing. Each has a nucleotide reiteration leading to a +1 frameshift and a nonsense codon downstream. The location of one frameshift mutation provided evidence that the first ATG is used for initiation of protein synthesis.

Amino Acid Sequence↗
PubMed1982

Sequence homologies of diverse length tandem repetitions near ends of vaccinia virus genome suggest unequal crossing over.

The 180,000 base pair (bp), covalently closed, linear duplex DNA genome of vaccinia virus contains a 10,000 bp inverted terminal repetition within which are one set of 13 and one set of 18 tandem 70 bp repeating units. A 967 bp segment containing the innermost 70 bp repeat and an adjacent region notable for a scarcity of restriction endonuclease sites has been sequenced. This was facilitated by the cloning of TaqI and partial TaqI fragments in pBR322. We found that the innermost 70 bp repeat overlaps one of two adjacent 125 bp repeats, following which are eight repeats of 54 bp, parts of 54 bp and 70 bp repeats, and four consecutive 6 to 7 bp repeats. The 70, 125, and 54 bp repeating units have extensive sequence homologies and redundancies that suggest evolution by unequal crossing over. Schemes whereby unequal crossovers of 54 bp repeats lead to a recombinant segment 86% homologous to the 125 bp repeat and unequal crossovers of 125 bp repeats lead to a recombinant segment 94% homologous to the 70 bp repeat were considered. This propensity for sequence divergence should provide a useful marker for comparing the relatedness of poxviruses.

Base Composition↗
PubMed1982

Purification and characterization of the messenger ribonucleic acid capping enzyme GTP:RNA guanylyltransferase from wheat germ.

A GTP:RNA guanylyltransferase or capping enzyme has been purified approximately 2000-fold from wheat germ. The enzyme catalyzes the transfer of the GMP residue from GTP to the 5' end of RNA or synthetic polyribonucleotides. Diphosphate-ended polymers were capped more efficiently than molecules with triphosphate ends, and molecules with monophosphate ends were not capped at all. There appears to be little specificity since RNAs with purine or pyrimidine ends served as acceptors. Other features of the wheat germ RNA guanylyltransferase include relatively low Km values for GTP (2.7 microM) and ppA (pA)n (14.2 nM), a divalent cation requirement satisfied by low (0.5 mM) concentrations of MnCl2 or higher (5 mM) concentrations of MgCl2, and a pH optimum around neutrality.

Kinetics↗
PubMed1982

Incompletely base-paired flip-flop terminal loops link the two DNA strands of the vaccinia virus genome into one uninterrupted polynucleotide chain.

The nature of the ends of the vaccinia virus genome was determined by nucleotide sequencing. Our finding of terminal hairpins indicated that the linear double-stranded DNA molecule consists of a single continuous polynucleotide chain. The 104 nucleotide apex of the hairpin contains predominantly A and T residues and is incompletely based-paired. These loops exist in two forms, which when inverted with respect to each other are complementary in sequence. Both forms of the 104 nucleotide loop are present in nearly equimolar amounts of each end of the genome. A set of 13 tandem 70 bp repeats begins 87 bp from the proximal segment of the terminal loop, followed by a unique sequence of 325 bp, and then by a second set of 18 tandem 70 bp repeats. The sequence of the 70 bp repeats reveals a 13 bp internal redundancy. Self-priming and de novo start replication models, which involve a site-specific nick in one DNA strand proximal to the 104 nucleotide loop, account for the observed sequence inversions and incomplete base-pairing. Similar mechanisms may be involved in replication of the ends of the eucaryotic chromosome.

Base Sequence↗
PubMed1982

Eukaryotic mRNA capping enzyme-guanylate covalent intermediate.

Incubation of HeLa cell mRNA guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) with [alpha-32P]GTP and a divalent cation in the absence of an RNA acceptor results in the formation of a covalent enzyme-guanylate complex. The complex, after purification by phosphocellulose chromatography, can transfer its bound GMP moiety to pyrophosphate, regenerating GTP, or to the 5'-diphosphate end of poly(A), forming a cap structure G(5')pppA(pA)n. The GMP-polypeptide has a molecular weight of 65,000 and is stable to heating in the presence of sodium dodecyl sulfate. On the basis of the alkali-stable and acid-labile nature of the bond and its susceptibility to nucleophilic attack by hydroxylamine at low pH, the GMP-polypeptide linkage appears to be a phosphoamine bond. After digestion with trypsin, a single GMP-peptide was resolved by two dimensional electrophoresis and chromatography.

Guanine Nucleotides↗
PubMed1982

Vaccinia virus: a selectable eukaryotic cloning and expression vector.

Foreign DNA was inserted into two nonessential regions of the vaccinia virus genome by homologous recombination in cells infected with virus and transfected with plasmids containing the foreign DNA elements flanked by vaccinia virus DNA. Thymidine kinase-negative (TK-) recombinants were selected after inserting foreign DNA into the coding region of the TK gene of wild-type vaccinia virus; TK+ recombinants were selected after inserting the herpesvirus TK gene into TK- mutants of vaccinia virus. For TK+ expression, it was necessary to insert a 275-base-pair DNA fragment containing the initiation site and sequences upstream of an early vaccinia virus transcript next to the coding sequences of the herpesvirus gene. The unique ability of the herpesvirus TK to phosphorylate 125I-labeled deoxycytidine provided independent confirmation of gene expression. These studies demonstrate the use of vaccinia virus as a selectable cloning and expression vector, confirm the map location of the vaccinia virus TK gene, and provide initial information regarding the location of vaccinia virus transcriptional regulatory sequences.

Cloning, Molecular↗
PubMed1982

Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA.

A selective plaque assay that uses thymidine kinase (TK)-deficient human 143 cells was developed to titer mixtures of TK(+) and TK(-) vaccinia virus. With this assay it could be shown that methotrexate-resistant TK(+) virus was formed in cells coinfected with TK(-) virus and wild-type virus DNA. By substituting vaccinia DNA fragments cloned in plasmids for virion DNA, this marker rescue system provided the basis for mapping the TK gene. Of the 15 HindIII fragments, only J could rescue five independently derived TK(-) mutants. This 5000-base-pair (bp) fragment maps approximately 80,000 bp from the left-end of the 180,000-bp vaccinia genome. Marker rescue could be detected with 18 ng or less of plasmid and was proportionate to DNA concentration. The resistance to methotrexate of the TK(+) recombinants was shown to be due to TK synthesis. Evidence that the HindIII J fragment contains the structural TK gene and not a regulatory element was demonstrated by the synthesis of active TK in a cell-free system programmed with mRNA selected by hybridization to the plasmid. Previous studies [Belle-Isle, H., Venkatesan, S. & Moss, B. (1981) Virology 112, 306-317] indicated that mRNAs coding for three immediate early polypeptides with molecular weights of 41,000, 21,000, and 17,000 map within HindIII J. The mapping of the easily selectable vaccinia virus TK gene now opens the way to genetic manipulations that should increase our understanding of vaccinia virus gene expression and facilitate the use of vaccinia virus as an efficient cloning vector for foreign genes.

Cell Line↗
PubMed1982

Colinearity of RNAs with the vaccinia virus genome: anomalies with two complementary early and late RNAs result from a small deletion or rearrangement within the inverted terminal repetition.

The colinearity of RNA transcripts with the vaccinia virus genome was investigated. Cytoplasmic RNA from infected cells was annealed to a cloned DNA segment that extended from 9 to 15.6 kilobase pairs from the left end of the genome and contained approximately 800 base pairs of the inverted terminal repetition (ITR). Remaining unhybridized single strands of DNA were digested with nuclease S1, and the lengths of the protected DNA fragments were determined by agarose gel electrophoresis under neutral and alkaline conditions. Uniformly 32P-labeled cloned DNA insert, separated recombinant DNA strands, and smaller restriction fragments, as well as 3' and 5' end-labeled DNA, were employed to map five early RNAs and one late RNA. One of the early RNAs hybridized to sequences within the ITR, and the other four hybridized to sequences proximal to the ITR. The late RNA was initiated proximal to the ITR but extended into it. Interestingly, the 3' portion of this late RNA was complementary to the early RNA transcribed from the opposite strand of the ITR. From a comparison of the lengths of the protected DNA fragments on neutral and alkaline gels, all except the complementary early and late RNAs appeared to be colinear with the genome. Although the anomalous nuclease S1 data obtained with the latter RNAs mimicked splicing, they were shown by DNA-DNA hybridization to result from a small deletion or rearrangement within the ITR. Thus far, no true examples of spliced vaccinia virus RNAs have been found.

Base Sequence↗
PubMed1982

Genetic evidence for vaccinia virus-encoded DNA polymerase: isolation of phosphonoacetate-resistant enzyme from the cytoplasm of cells infected with mutant virus.

Phosphonoacetate (PAA), at concentrations of 200 micrograms/ml or more, prevented growth of vaccinia virus in HeLa and BSC-1 cells. Spontaneous vaccinia virus mutants, selected at high PAA levels, were resistant to the antiviral effects of the drug. The action of PAA was directed toward an early viral function, since the drug was inhibitory only during the first 4 h of the approximately 15-h growth cycle. Conversely, significant reversal of the antiviral effects was obtained only when the drug was removed at or before the fourth hour of infection. Incorporation of [3H]thymidine into cytoplasmic viral DNA was severely inhibited in cells infected with wild-type virus but not in cells infected with mutant virus. Virus-induced DNA polymerase isolated from the cytoplasm of cells infected with wild-type or mutant virus had indistinguishable chromatographic properties on DEAE-cellulose and phosphocellulose columns. However, the wild-type enzyme was inhibited by relatively low concentrations of PAA, whereas 10-fold higher concentrations were needed for equivalent inhibition of the mutant enzyme. Kinetic analysis indicated that PAA inhibition was noncompetitive with deoxyribonucleoside triphosphates; Ki values for wild-type and mutant DNA polymerases were approximately 25 and 300 microM, respectively. Inhibition of wild-type DNA polymerase was immediate and complete even when PAA was added after initiation of DNA synthesis in vitro, suggesting that chain elongation was affected. These results established that the DNA polymerase is a target of the antiviral action of PAA and provided genetic evidence that this enzyme is virus encoded.

Animals↗
PubMed1982

Complete nucleotide sequences of two adjacent early vaccinia virus genes located within the inverted terminal repetition.

The proximal part of the 10,000-base pair (bp) inverted terminal repetition of vaccinia virus DNA encodes at least three early mRNAs. A 2,236-bp segment of the repetition was sequenced to characterize two of the genes. This task was facilitated by constructing a series of recombinants containing overlapping deletions; oligonucleotide linkers with synthetic restriction sites provided points for radioactive labeling before sequencing by the chemical degradation method of Maxam and Gilbert (Methods Enzymol. 65:499-560, 1980). The ends of the transcripts were mapped by hybridizing labeled DNA fragments to early viral RNA and resolving nuclease S1-protected fragments in sequencing gels, by sequencing cDNA clones, and from the lengths of the RNAs. The nucleotide sequences for at least 60 bp upstream of both transcriptional initiation sites are more than 80% adenine . thymine rich and contain long runs of adenines and thymines with some homology to procaryotic and eucaryotic consensus sequences. The gene transcribed in the rightward direction encodes an RNA of approximately 530 nucleotides with a single open reading frame of 420 nucleotides. Preceding the first AUG, there is a heptanucleotide that can hybridize to the 3' end of 18S rRNA with only one mismatch. The derived amino acid sequence of the protein indicated a molecular weight of 15,500. The gene transcribed in the leftward direction encodes an RNA 1,000 to 1,100 nucleotides long with an open reading frame of 996 nucleotides and a leader sequence of only 5 to 6 nucleotides. The derived amino acid sequence of this protein indicated a molecular weight of 38,500. The 3' ends of the two transcripts were located within 100 bp of each other. Although there are adenine . thymine-rich clusters near the putative transcriptional termination sites, specific AATAAA polyadenylic acid signal sequences are absent.

Amino Acid Sequence↗