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J A Esteban

Publications and source records attributed to J A Esteban.

35 records · Page 2Linked to original sources
PubMed1992

Initiation of phi 29 DNA replication occurs at the second 3' nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication.

Bacteriophage phi 29 DNA replication is initiated when a molecule of dAMP is covalently linked to a free molecule of the terminal protein, in a reaction catalyzed by the viral DNA polymerase. We demonstrate that single-stranded DNA molecules are active templates for the protein-primed initiation reaction and can be replicated by phi 29 DNA polymerase. Using synthetic oligonucleotides, we carried out a mutational analysis of the phi 29 DNA right end to evaluate the effect of nucleotide changes at the replication origin and to determine the precise initiation site. The results indicate that (i) there are no strict sequence requirements for protein-primed initiation on single-stranded DNA; (ii) initiation of replication occurs opposite the second nucleotide at the 3' end of the template; (iii) a terminal repetition of at least two nucleotides is required to efficiently elongate the initiation complex; and (iv) all the nucleotides of the template, including the 3' terminal one, are replicated. A sliding-back model is proposed in which a special transition step from initiation to elongation can account for these results. The possible implication of this mechanism for the fidelity of the initiation reaction is discussed. Since all the terminal protein-containing genomes have some sequence reiteration at the DNA ends, this proposed sliding-back model could be extrapolable to other systems that use proteins as primers.

Bacillus Phages↗
PubMed1992

The bacteriophage phi 29 DNA polymerase, a proofreading enzyme.

The bacteriophage phi 29 DNA polymerase, involved both in the protein-primed initiation and elongation steps of the viral DNA replication, displays a very processive 3',5'-exonuclease activity acting preferentially on single-stranded DNA. This exonucleolytic activity showed a marked preference for excision of a mismatched versus a correctly paired 3' terminus. These characteristics enable the phi 29 DNA polymerase to act as a proofreading enzyme. A comparative analysis of the wild-type phi 29 DNA polymerase and a mutant lacking 3',5'-exonuclease activity indicated that a productive coupling between the exonuclease and polymerase activities is necessary to prevent fixation of polymerization errors. Based on these data, the phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication, appears to share the same mechanism for the editing function as that first proposed for T4 DNA polymerase and Escherichia coli DNA polymerase I on the basis of functional and structural studies.

Bacteriophages↗
PubMed1992

Metal activation of synthetic and degradative activities of phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication.

Analysis of metal activation on the synthetic and degradative activities of phi 29 DNA polymerase was carried out in comparison with T4 DNA polymerase and Escherichia coli DNA polymerase I (Klenow fragment). In the three DNA polymerases studied, both the polymerization and the 3'----5' exonuclease activity had clear differences in their metal ion requirements. The results obtained support the existence of independent metal binding sites for the synthetic and degradative activities of phi 29 DNA polymerase, according with the distant location of catalytic domains (N-terminal for the 3'----5' exonuclease and C-terminal for DNA polymerization) proposed for both Klenow fragment and phi 29 DNA polymerase. Furthermore, DNA competition experiments using phi 29 DNA polymerase suggested that the main differences observed in the metal usage to activate polymerization may be the consequence of metal-induced changes in the enzyme-DNA interactions, whose strength distinguishes processive and nonprocessive DNA polymerases. Interestingly, the initiation of DNA polymerization using a protein as a primer, a special synthetic activity carried out by phi 29 DNA polymerase, exhibited a strong preference for Mn2+ as metal activator. The molecular basis for this preference is mainly the result of a large increase in the affinity for dATP.

Binding Sites↗
PubMed1992

DNA-independent deoxynucleotidylation of the phi 29 terminal protein by the phi 29 DNA polymerase.

In this paper, we show that the phi 29 DNA polymerase, in the absence of DNA, is able to catalyze the formation of a covalent complex between the phi 29 terminal protein (TP) and 5'-dAMP. Like the reaction in the presence of phi 29 DNA, TP.dAMP complex formation is strongly dependent on activating Mn2+ ions and on the efficient formation of a TP/DNA polymerase heterodimer. The nature of the TP-dAMP linkage was shown to be identical (a O-5'-deoxyadenylyl-L-serine bond) to that found covalently linking TP to the DNA of bacteriophage phi 29, indicating that this DNA-independent reaction actually mimics that occurring as the initiation step of phi 29 DNA replication. Furthermore, as in normal TP-primed initiation on the phi 29 DNA template, this novel reaction showed the same specificity for TP Ser232 as the OH donor and the involvement of the YCDTD amino acid motif, highly conserved in alpha-like DNA polymerases. However, unlike the reaction in the presence of phi 29 DNA, the DNA-independent deoxynucleotidylation of TP by the phi 29 DNA polymerase did not show dATP specificity, being possible to obtain any of the four TP.dNMP complexes with a similar yield. This lack of specificity together with the poor efficiency of this reaction at low deoxynucleoside triphosphate (dNTP) concentration reflect a weak, but similar stability of the four dNTPs at the phi 29 DNA polymerase dNTP-binding site. Thus, the presence of a director DNA would mainly contribute to stabilizing a complementary nucleotide, giving base specificity to the protein-primed initiation reaction. According to all these data, the novel DNA polymerase reaction described in this paper could be considered as a "non-DNA-instructed" protein-primed deoxynucleotidylation.

Bacteriophages↗
PubMed1992

Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase; overlapping structural domains for the 3'-5' exonuclease and strand-displacement activities.

In this report we present the alignment of one of the most conserved segments (Exo III) of the 3'-5' exonuclease domain in 39 DNA polymerase sequences, including prokaryotic and eukaryotic enzymes. Site-directed substitutions of the two most conserved residues, which form the Exo III motif Tyr-(X)3-Asp of phi 29 DNA polymerase, did not affect single-stranded DNA binding, DNA polymerization, processivity or protein-primed initiation. In contrast, substitution of the highly conserved Tyr residue by Phe or Cys decreased the 3'-5' exonuclease activity to 7.5 and 4.1%, respectively, of the wild-type activity. Change of the highly conserved Asp residue into Ala resulted in almost complete inactivation (0.1%) of the 3'-5' exonuclease. In accordance with the contribution of the 3'-5' exonuclease to the fidelity of DNA replication, the three mutations in the Exo III motif (Y165F, Y165C and D169A) produced enzymes with an increased frequency of misinsertion and extension of DNA polymerization errors. Surprisingly, the three mutations in the Exo III motif strongly decreased (80- to 220-fold) the ability to replicate phi 29 DNA, this behaviour being due to a defect in the strand displacement activity, an intrinsic property of phi 29 DNA polymerase required for this process. Taking these results into account, we propose that the strand displacement activity of phi 29 DNA polymerase resides in the N-terminal domain, probably overlapping with the 3'-5' exonuclease active site.

Amino Acid Sequence↗
PubMed1992

Structural and functional studies on phi 29 DNA polymerase.

The Bacillus subtilis phage phi 29 DNA polymerase, involved in protein-primed viral DNA replication, contains several amino acid consensus sequences common to other eukaryotic-type DNA polymerases. Using site-directed mutagenesis, we have studied the functional significance of a C-terminal conserved region, represented by the Lys-X-Tyr ("K-Y") motif. Single point mutants have been constructed and the corresponding proteins have been overproduced and characterized. Measurements of the activity of the mutant proteins indicated that the invariant Lys and Tyr residues play a critical role in DNA polymerization. Interestingly, substitution of the invariant Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respectively, capability to use a protein as primer, an intrinsic property of TP-priming DNA polymerases. On the other hand, the viral protein p6, which stimulates initiation of phi 29 DNA replication by formation of a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity of phi 29 DNA polymerase during the formation of the TP-dAMP initiation complex. We propose a model in which the special strategy to maintain the integrity of the phi 29 DNA ends, by means of a "sliding-back" mechanism, could also contribute to increase the fidelity of phi 29 DNA replication.

Amino Acid Sequence↗
PubMed1992

Mechanical complications of the reservoirs and flushing devices in ventricular shunt systems.

Complications of valve reservoirs or flushing devices are seldom reported as a cause of shunt malfunction. The authors document their experience in six cases of mechanical complications of shunting devices. Two cases presented with collapse and intracranial migration of the valve reservoir, a complication that has not been reported previously. In two cases, the plastic dome became disconnected from the metallic base of the reservoir. A fifth patient, with an integral shunt system, showed a fracture in the soldered join of the distal tube to the reservoir dome. The last patient had the valve reservoir partially collapsed by bone growth at the time of shunt revision.

Cerebrospinal Fluid Shunts↗
PubMed1991

Meningiomas with haemorrhagic onset.

Classically meningiomas present and evolve with a progressive course. Meningiomas manifest themselves mainly in middle and old age when the incidence of strokes is higher. The authors report three instances of meningioma with acute haemorrhagic onset. The clinical picture of our cases consisted of subarachnoid bleeding, subdural hematoma and intracerebral haemorrhage respectively. The diagnostic difficulties encountered when assessing these patients are discussed. The current literature regarding meningioma associated with haemorrhage is reviewed. One of our cases seems to be the first reported case of subarachnoid haemorrhage originating from a meningioma of the petrous bone.

Aged↗
PubMed1991

PASHEMOS: a versatile program written in Pascal to simulate patterns on the shells of molluscs, according to the Meinhardt model.

In this paper we describe PASHEMOS, a user-friendly program written in Pascal to solve the Meinhardt-Klingler model numerically. This model studies the shell pattern pigmentation of molluscs. Moreover, PASHEMOS enables theoretical analysis to be performed on the different steady states. The program takes into account the four differential equation systems established in the model. It numerically solves these systems. This program is developed in such a way that both the equation system type and the parameter values can be chosen. The pattern is displayed in each case, and other output possibilities are available (i.e. three-dimensional concentration plotting, phase plane, phase space, stability analysis). The implementation of the program for the case of a three-variable system is shown as an example of PASHEMOS handling. Program applications for other models are also discussed.

Algorithms↗
PubMed1990

Cloning and characterization of the gene encoding a blasticidin S acetyltransferase from Streptoverticillum sp.

A gene (bls) encoding a blasticidin S-acetyltransferase from Streptoverticillum sp. JCM4673 was cloned in the Streptomyces plasmid pIJ702 and expressed in S. lividans 1326. Four of the recombinant plasmids contained a common 1129-bp BclI-PstI DNA fragment, which by subcloning experiments was shown to carry the bls gene. Sequencing of the 1129-bp BclI-PstI fragment indicated that it contains two open reading frames of 408 and 366 nucleotides (nt), both starting with ATG and terminating at TGA triplets. Deletion analysis showed that the correct bls sequence is that of 408 nt, whose deduced amino acid sequence conforms to a polypeptide of Mr 15073. A 175-bp DNA fragment from the 5' region of the bls gene had promoter activity in the promoter-probe plasmid pIJ486.

Acetyltransferases↗
PubMed1987

Astrocytoma of the cerebral aqueduct.

The authors report the case of a 9-year-old girl with a low-grade astrocytoma of the mesencephalon that occluded the sylvian aqueduct. Symptoms, signs, and neuroradiological investigations led to the clinical impression of aqueductal stenosis. The presence of a tumor was diagnosed by cerebrospinal fluid cytology and confirmed at postmortem examination. This is the first reported instance of this tumor being diagnosed by cerebrospinal fluid cytology and the third report of a diagnosis in a living patient. The authors stress the value of CSF cytology in evaluating a typical case of hydrocephalus.

Astrocytoma↗
PubMed1986

Subarachnoid hemorrhage in the presence of a cerebral arteriovenous malformation and an intraventricular oligodendroglioma: case report.

The authors report a case of a ventricular oligodendroglioma associated with a cortical arteriovenous malformation. The patient presented with subarachnoid hemorrhage. Computed tomographic scan showed an intraventricular hyperdense lesion, mimicking a hematoma. Angiography revealed a superficial arteriovenous malformation in the right parietal lobe, unrelated to ventricular cavities. Both lesions were treated during the same operation. The diagnostic difficulties and the significance of this uncommon association are discussed.

Adult↗
PubMed1985

Intracranial tumor biopsy--CT-guided stereotactic surgery.

A modification of Gildenberg's technique for brain tumor biopsy is described. Marking the light beam of the gantry on the scalp with a pencil, when the lesion appears on the screen, no ScoutView is necessary. With radiopaque marks on the drawn lines, the levels of the slice are transferred to a lateral conventional X-ray, for calculation of the 'Z' coordinate. 'X' and 'Y' coordinates are determined on the CT scanner.

Adolescent↗
PubMed1984

Craniostenosis secondary to calcified subperiosteal hematoma: case report.

The case of a 5-month-old boy with sagittal synostosis secondary to a calcified cephalohematoma of the cranial vault is reported. Although birth trauma has been suggested as a cause of craniosynostosis, a review of the current literature failed to show a similar case. Surgical treatment to remove the calcified hematoma produced good cosmetic and functional results.

Calcinosis↗
PubMed1981

[Treatment of peritoneal hydatidosis with mebendazole. Preliminary study (author's transl)].

Authors present three cases of peritoneal hydatidosis, two of which are due to spontaneous rupture of hepatic hydatid cysts and the other due to operative rupture of ovarian hydatid cyst. A dose of 1 g./day of mebendazole was administered for 11 months with 30 day break periods every three months. Mebendazole has proved effective in the treatment of secondary echinococcosis. In authors judgement this may be an effective treatment for peritoneal spreading if mebendazole is used at an early stage since they have verified the absence of dissemination in one of the cases and an inactive dissemination in other one. If treatment is applied later, regression is achieved too, but more slowly. Immunological and ecographical results are reported in detail as well as surgical and pathologic findings. A cautious interpretation of these preliminary results is advised since data on long-term results are lacking.

Benzimidazoles↗