Bacteriophage ø29 DNA polymerase shares with other DNA-dependent DNA polymerases several regions of amino acid homology along the primary structure. Among them, motif B, characterized by the consensus +x3Kx(6-7)YG (+ being a positively charged amino acid), appears to be specifically conserved in those polymerases that use DNA but not RNA as template. In particular, the lysine residue of this motif is invariant in all members of DNA-dependent polymerases.
View Article and Find Full Text PDFPhage phi29 from Bacillus subtilis is a paradigm of the protein-primed replication mechanism, in which a single-subunit DNA polymerase is involved in both the specific protein-primed initiation step and normal DNA elongation. To start phi29 DNA replication, the viral DNA polymerase must interact with a free molecule of the viral terminal protein (TP), to prime DNA synthesis once at each phi29 DNA end. The results shown in this paper demonstrate that the DNA polymerase-primer TP heterodimer is not dissociated immediately after initiation.
View Article and Find Full Text PDFBy using a baculovirus expression system, the adenovirus (Ad) DNA polymerase was purified to homogeneity and shown to display a 3'-->5'exonuclease activity which is coupled to the polymerase activity. On a partial duplex structure the exonuclease activity had a marked preference for excision of a mismatched versus a matched 3'-terminus, which enables the Ad DNA polymerase to act as a proofreading enzyme. On single-stranded DNA the exonuclease action is distributive, but during replication removal of mismatched nucleotides and the switch to synthesis occurs without dissociation of the polymerase from the template.
View Article and Find Full Text PDFA new self-sustained terminal protein-primed DNA amplification system has been used to describe in vitro evolutionary changes affecting maintenance of the genome size of bacteriophage phi29. These changes involve generation and efficient amplification of short palindromic molecules containing an inverted duplication of one of the original DNA ends. A template-switching mechanism is proposed to account for the appearance of these molecules.
View Article and Find Full Text PDFWe have obtained a clinical pregnancy in a 26 year old patient by means of in vitro oocyte fertilization by intracytoplasmic injection of spermatozoa obtained from a cryopreserved testicular biopsy. In a first attempt performed with fresh biopsy material, the woman became pregnant, but the pregnancy ended in a spontaneous abortion. In a second cycle, the spermatozoa were retrieved from a cryopreserved sample saved from the first attempt.
View Article and Find Full Text PDFMicrobiol Immunol
April 1998
The immunogenic effect of Salmonella typhi OmpC porin during typhoid fever in humans was evaluated in vitro. Peripheral blood mononuclear cells from 17 patients were challenged with outer membrane preparations from Escherichia coli UH302 and UH302/pSTP2K2 strains, both lacking E. coli OmpF and OmpC porins, although UH302/pSTP2K2 expressed a plasmid-encoded S.
View Article and Find Full Text PDFWe have determined the nucleotide sequence of 2676 bp at the left part of the linear genome of Bacillus subtilis bacteriophage GA-1. Computer analysis revealed that this fragment contains two open reading frames (ORFs), ORF1 and ORF2, which contain 265 and 578 codons, respectively. Data base search revealed that ORF1 and ORF2 might encode proteins similar to the terminal protein (TP) and DNA polymerase, respectively, of bacteriophage phi29.
View Article and Find Full Text PDFOral Surg Oral Med Oral Pathol Oral Radiol Endod
November 1996
Thirty permanent incisors with vital pulps and complicated crown fractures were treated by a partial pulpotomy (Cvek technique). This consisted of amputation of 1 to 2 mm of the exposed pulp, placement of calcium hydroxide powder, and a temporary restoration. Clinical and radiographic assessment of the hard tissue barrier was done after 3 months and again after 1 to 8 years.
View Article and Find Full Text PDFPhage Cp-1 from Streptoccocus pneumoniae makes use of a protein-priming mechanism to start replication of its linear DNA: the first reaction consists of the addition of 5' dAMP to a molecule of the primer protein, an initiation event occurring at both DNA ends. After elongation of the initiation complex, the primer protein remains linked to the 5' end of the nascent DNA chain, and is subsequently referred to as terminal protein (TP). In this paper, using DNA-free extracts from Cp-1-infected S.
View Article and Find Full Text PDFThe functional significance of the conserved motif 'YxGG/A', located between the 3'-5' exonuclease and polymerization domains of eukaryotic-type DNA polymerases, has been studied by site-directed mutagenesis in phi29 DNA polymerase. Single substitutions at this region were obtained, and 11 phi29 DNA polymerase mutant derivatives were overproduced in Escherichia coli and purified to homogeneity. Nine mutants showed an altered polymerase/3'-5' exonuclease balance on a template/primer DNA structure, giving rise to three different mutant phenotypes: (i) favored polymerization (high pol/exo ratio); (ii) favored exonucleolysis (low pol/exo ratio); and (iii) favored exonucleolysis and null polymerization.
View Article and Find Full Text PDFWe have now applied the enzyme immunoassay using anti-NGF monoclonal antibody (MAb) 27/21 and a blocking test validating the specificity of the immunoreactivity for NGF in serum samples to examine NGF levels in normal rat sera, hemiparkinsonian rat sera, normal monkey sera, and MPTP-treated monkey sera. The levels of NGF in treated animals showed reductions when compared with serum from normal animals. The NGF level alterations observed in lesioned animals and in human parkinsonian patients evidence a relationship between this neurotrophic factor and the neurodegenerative changes observed in Parkinson disease (PD).
View Article and Find Full Text PDFBy site-directed mutagenesis in phi29 DNA polymerase, we have analyzed the functional importance of two evolutionarily conserved residues belonging to the 3'-5' exonuclease domain of DNA-dependent DNA polymerases. In Escherichia coli DNA polymerase I, these residues are Thr358 and Asn420, shown by crystallographic analysis to be directly acting as single-stranded DNA (ssDNA) ligands at the 3'-5' exonuclease active site. On the basis of these structural data, single substitution of the corresponding residues of phi29 DNA polymerase, Thr15 and Asn62, produced enzymes with a very reduced or altered capacity to bind ssDNA.
View Article and Find Full Text PDFTo study the effect of prolonged diabetes on protein synthesis and on the activities of initiation factors eIF-2 and eIF-2B in the liver, female rats were treated with streptozotocin. Some animals were mated and studied on day 20 of pregnancy, whereas others were kept virgin and studied in parallel. The protein synthesis rate was measured with an "in vitro' cellfree system, and was lower in diabetic pregnant and virgin animals than in pregnant and virgin controls (30-60%).
View Article and Find Full Text PDFAmino acids Tyr254 and Tyr390 of phi 29 DNA polymerase belong to one of the most conserved regions in eukaryotic-type DNA polymerases. In this paper we report a mutational study of these two residues to address their role in nucleotide selection. This study was carried out by means of a new kinetic analysis that takes advantage of the competition between DNA polymerization and 3'-->5' exonuclease activity to measure the Km values for correct and incorrect nucleotides in steady-state conditions.
View Article and Find Full Text PDFDuring development, rat lung seems to make use of the mechanisms of 1) expansion, 2) replication, and 3) subdivision to increase its gas-exchange surface area. To quantitate the contribution of each one of these mechanisms from birth to adulthood, four models of lung development have been mathematically evaluated: 1) existing saccules at birth septate (subdivide) during the first 2 postnatal weeks, creating smaller alveoli that undergo a continuous expansion up to adulthood; 2) same as the previous model except that new alveoli are also formed by means other than septation after the second postnatal week; 3) same as the previous model except that the alveoli formed by means other than septation start forming immediately after birth; and 4) saccules continue to be formed up to adulthood and subsequently septate, forming alveoli. In an evaluation of these models mathematically, it was found that expansion followed by replication is the most important mechanism for the generation of gas-exchange surface area in all four models in the maturing lung.
View Article and Find Full Text PDFFEMS Microbiol Rev
August 1995
phi 29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the phi 29 DNA ends.
View Article and Find Full Text PDFToxic shock syndrome toxin 1 (TSST-1), a superantigen produced by Staphylococcus aureus, is a causative agent of toxic shock syndrome (TSS). This superantigen is a potent stimulator of T cells and macrophages/monocytes, resulting in the release of cytokines that are implicated in the pathogenesis of TSS. This study characterizes a mutant TSST-1, derived by site-directed mutagenesis, that has an alanine substitution at histidine 135 (mutant 135).
View Article and Find Full Text PDFphi 29 DNA polymerase shares with other DNA-dependent DNA polymerases several regions of amino acid homology along the primary structure. A conserved amino acid motif, located in the C-terminal portion of the polypeptide and characterized by the amino acid sequence KK(K/R)Y, is conserved in the group of eukaryotic-type DNA polymerases. In the subgroup of DNA polymerases that have a protein-priming mechanism, this motif is restricted to the sequence KXY, X never being a positively charged amino acid.
View Article and Find Full Text PDFTo study the effect of diabetes on hepatic protein synthesis and polysomal aggregation in pregnant rats, female rats were treated with streptozotocin prior to conception. Some animals were mated, and studied at day 20 of pregnancy, whereas, others were studied in parallel under non pregnant conditions. The protein synthesis rate measured with an "in vitro" cell-free system was higher in pregnant than in virgin control rats.
View Article and Find Full Text PDFThe residues forming the 3'-->5' exonuclease active site of phi 29 DNA polymerase, located at the N-terminal conserved motifs Exo I, Exo II and Exo III, have been defined by site-directed mutagenesis (Bernad, A., Blanco, L., Lázaro, J.
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