Biomedical subjects

B M Baroudy

Publications and source records attributed to B M Baroudy.

45 records · Page 3Linked to original sources

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

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↗

PubMed1981

Distinctive nucleotide sequences adjacent to multiple initiation and termination sites of an early vaccinia virus gene.

Poxviruses, unlike other DNA viruses, replicate in the cytoplasm of infected cells and use their own system of transcription. Examination on one early mRNA synthesized in vivo and in vitro indicated that it has multiple closely spaced 5' and 3' ends. A remarkable 88% AT-rich 60 bp DNA sequence was found immediately upstream of the initiation of transcription sites. Although DNA sequences that bear some homology to Pribnow and Hogness boxes are present, additional recognition sequences located further upstream of procaryotic and eucaryotic initiation sites are absent. A possible initiation of translation codon occurs about 50 nucleotides from the 5' end of the message. The transcript terminates near or within a hexanucleotide CTATTC that is tandemly repeated four times. Sequences similar to those regulating termination of transcription in procaryotes or poly (A) addition in eucaryotes were not found, suggesting that poxviruses have evolved unique recognition signals.

Cytoplasm↗

PubMed1980

Purification and characterization of a DNA-dependent RNA polymerase from vaccinia virions.

A DNA-dependent RNA polymerase has been extracted from vaccinia virions and purified to near homogeneity as judged by glycerol gradient sedimentation and polyacrylamide gel electrophoresis. The native enzyme has a molecular weight of approximately 500,000 and can be dissociated into putative subunits of 140,000, 137,000, 37,000, 35,000, 31,000, 22,000, and 17,000 daltons. Activity was dependent on all four ribonucleoside triphosphates, Mn2+, and a DNA template. Optimal activity occurred at pH 7.9 in the presence of 90 mM KCl or 40 mM (NH4)2SO4. All single-stranded DNAs tested served as templates. By contrast, linear double-stranded DNAs were not effectively transcribed and very low activity was obtained with circular supercoiled DNAs which contain small single-stranded regions. The synthetic alternating copolymer poly(dA-dT), which forms a completely base-paired structure, also was not transcribed, whereas poly(dA,dT) and other random copolymers served as templates. Of the four homopolydeoxribonucleotides, only poly(dC) and poly(dT) were transcribed, suggesting that initiation specifically occurs with purine ribonucleotides. In support of this, higher Km values were obtained for GTP and ATP (333 and 80 micro M, respectively) than for UTP and CTP (22 and 12 micro M, respectively) using a DNA template. The RNA polymerase was inhibited by polyanions but was resistant to rifampicin and alpha-amanitin.

Base Composition↗

PubMed1980

Structural requirements for binding of bovine tRNATrp with avian myeloblastosis virus DNA polymerase.

Avian DNA polymerases use host tRNATrp as the primer for transcription. Bovine tRNATrp has been previously shown to be a biologic substitute for the avian primer. A bovine tRNATrp fragment has been identified as having a high binding affinity for the polymerase. The fragment is assigned to be 67 nucleotides, and contains most of the elements required to maintain the secondary and tertiary structure of tRNATrp.

Animals↗

PubMed1977

Magnesium ion requirements for yeast enolase activity.

It has generally been concluded that two divalent cations are required for enolase activity, even though the enzyme is a homodimer that specifically binds four metal ions in the presence of substrate. This paper reports a reinvestigation of the stoichiometry of enolase activation. Specific ion electrode measurements of Mg2+ binding in the presence and absence of substrate are compared with stopped-flow measurements of the velocity of 2-phosphoglycerate dehydration. It is concluded that the enzyme is inactive when only two metal-binding sites are filled and that four sites must be populated with Mg2+ for full activity. An ordered binding mechanism is proposed that quantitatively predicts the activation of enolase by the four Mg2+ ions from their measured dissociation constants and the Michaelis constant for the dehydration reaction. To explain the loss of enzymatic activity at still higher metal concentrations, the binding of additional, inhibitory Mg2+ ions is postulated.

Binding Sites↗

PubMed1977

tRNATrp (bovine) binding to the reverse transcriptase of avian myeloblastosis virus and function as a heterologous primer.

The primary structures for tTNATrp (bovine) and primer tRNATrp (avian) show only minor differences in nucleotide sequence. The heterologous tRNATrp (bovine) appears to have properties similar to the tRNATrp (avian) in its ability to bind the alphabeta from of RNA-dependent DNA nucleotidyltransferase of avian myeloblastosis virus. A stable enzyme-tRNA complex has been isolated by gel filtration. In addition, tRNATrp (bovine) can hydridize to the avian viral 35S RNA and act as a primer for transcription of the RNA. tRNATrp (bovine) can be obtained in larger amounts than the avian primer and can be used to study the interactions between the primer and the viral enzyme.

Avian Leukosis Virus↗

PubMedDate not supplied

Application of nucleic acid hybridization to the hepatitis delta virus research.

Cloning and sequencing of the genome of hepatitis delta virus (HDV) represented a remarkable progress in the knowledge of HDV infection and made available cDNA and RNA probes which can be successfully used to identify HDV-RNA in biological specimens. We review here the methods currently used for detecting HDV nucleic acids and report the most recent advances of the molecular biology of HDV. Finally, we examine the number of compelling reasons for the diagnostic laboratory to pursue this technology. Testing for HDV-RNA has become essential for the screening and the follow-up of both chronic HDV carriers, undergoing antiviral therapy and patients with terminal disease, eligible for liver transplant.

Hepatitis D↗

PubMedDate not supplied

In situ hybridization: perspectives of its clinical application in hepatitis B and delta virus infections.

In situ hybridization is a sensitive and specific technique for detecting nucleic acids in cytological and histological specimens. Visualization of the target sequences by autoradiography or (immuno)histochemistry allows their precise subcellular localization and quantitation. The application of in situ hybridization techniques has contributed to the understanding of the complex replicative cycle of hepatitis B virus. More recently, hepatitis delta virus replication has also been studied by this technique. Although in situ hybridization is a powerful tool for the molecular biologist, its clinical significance in diagnosis and prognosis of human hepatitis virus infections has yet to be fully evaluated.

Hepatitis B↗