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Biomedical subjects

A Bravo

Publications and source records attributed to A Bravo.

At least 73 records · Page 4Linked to original sources
PubMed1994

DNA structure in the nucleoprotein complex that activates replication of phage phi 29.

Initiation of phage phi 29 DNA replication is activated by the viral protein p6 which forms a nucleoprotein complex at the replication origins, located at the linear genome ends. The complex consists of a DNA right-handed superhelix wrapped around a multimeric protein core. We have determined the superhelical path of the DNA in the complex, measuring the change in linking number induced by the protein, the surface-related helical repeat and the compaction of the DNA. One superhelical turn has approximately 63 bp (2.6 p6 dimers). Furthermore, we have determined that the DNA binding domain of protein p6 is located at the N-terminal region, predicted to form an amphipathic alpha-helix. We have obtained, by site-directed mutagenesis, protein p6 mutants in the polar side of the putative helix in which their DNA binding and replication activation properties were impaired or undetectable, in agreement with in vivo results.

Bacillus Phages↗
PubMed1994

Sequences 5' of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development.

Expression of keratin K5 (and K14) in multilayered epithelia occurs predominantly in the basal layer of proliferating keratinocytes. When a keratinocyte becomes committed to terminal differentiation, it moves out of the basal layer towards the epithelial surface. As part of this program of terminal differentiation, the expression of K5 (and K14) is downregulated in suprabasal cells, and new pairs of differentiation-specific keratins are expressed. To define the cis-acting DNA sequences required for K5 cell-type- and differentiation-specific expression, chimeric gene fusions between portions of the bovine keratin K5 locus and the Escherichia coli lacZ gene were used to generate transgenic mice. In the genomic fragment consisting of 5.3 kb of 5' flanking sequences, 6.1 kb corresponding to the body of the gene and 4.5 kb of 3' flanking sequences, the subfragment extending from -5300 bp to +138 bp was the smaller region that directed lacZ expression to stratified epithelia in a manner analogous to the endogenous keratin K5. Proximal sequences from -1300 bp to +138 bp were inactive. We also determined the expression pattern of keratin K5 during mouse development using an antiserum specific for mouse keratin K5. Expression was first detected in ectodermal cells of 11.5 days postcoitum embryos, and from day 13.5 postcoitum onwards K5 was detected in the precursors of most epithelia and organs which express K5 at adult stages. This pattern was reproduced, with few differences, by the construct with sequences from -5300 bp to +138 bp fused to the lacZ gene. These findings identify sequences between -5.3 kb and -1.3 kb of the bovine K5 gene as being important for cell-type- and differentiation-specific gene expression both during mouse development and in the adult.

Aging↗
PubMed1994

Vectors of cutaneous leishmaniasis in north-central Venezuela.

An entomological survey was undertaken from January 1991 to February 1992 in El Ingenio, Miranda State, Venezuela, an endemic area of cutaneous leishmaniasis: prevalence of 10.7 cases per 100,000 inhabitants. A total of 4863 female sandflies (Phlebotominae) of fourteen species were collected in Shannon traps, then dissected and examined for leishmanial infections. Lutzomyia ovallesi (85.4%) and Lu. gomezi (11.2%) were the predominant anthropophilic species of sandfly. Fifty-one (1.19%) Lu.ovallesi and two (0.47%) Lu.gomezi had natural infection with Leishmania promastigotes. Identification of the parasites was done by using the polymerase chain reaction (PCR) and DNA hybridization. Two isolates from Lu.gomezi and forty-nine from Lu.ovallesi were typed as Leishmania braziliensis and three of the latter reacted with Le.mexicana also. This is the first report of Lu.gomezi with parasites typed as Le.braziliensis. We concluded that Lu.ovallesi is the primary vector of cutaneous leishmaniasis in the north-central area of Venezuela and Lu.gomezi should be regarded as an additional vector.

Animals↗
PubMed1994

PCR analysis of the cryI insecticidal crystal family genes from Bacillus thuringiensis.

A method allowing rapid and accurate identification of different subgroups within the insecticidal crystal CryI protein-producing family of Bacillus thuringiensis strains was established by using PCR technology. Thirteen highly homologous primers specific to regions within genes encoding seven different subgroups of B. thuringiensis CryI proteins were described. Differentiation among these strains was determined on the basis of the electrophoretic patterns of PCR products. B. thuringiensis strains, isolated from soil samples, were analyzed by PCR technology. Small amounts of bacterial lysates were assayed in two reaction mixtures containing six to eight primers. This method can be applied to rapidly detect the subgroups of CryI proteins that correspond with toxicity to various lepidopteran insects.

Bacillus thuringiensis↗
PubMed1994

[Adult T-cell lymphoma and Reed-Sternberg cells. A clinical case].

Adult T-cell lymphoma (ATL) is a neoplastic disease associated in 80% of cases with HTLV-I (Human T Lymphotropic virus type I) infection. 1-2% of HTLV-I carriers may develop the disease during their lives. In our country, the prevalence of HTLV-I infection is about 1%. We report a 72 y.o. woman HTLV-I positive, confirmed by Western-Blot. presenting with massive adenopathies, cutaneous tumor involvement, acute course and showing Reed-Sternberg cells (RSCs) in lymph node biopsy. The response to chemotherapy was poor, with the patient dying seven months after diagnosis. ATL and Hodgkin's disease (HD) are discussed highlighting that RSCs are not pathognomonic of HD though in their absence such diagnosis can not be made. RSCs are found in other benign and malignant disorders and it is especially important to differentiate HD from the other processes in order to dispense the appropriate treatment. This is the first case of ATL with RSCs reported in Chile. Hence, in our country we suggest systematic determination of the presence of HTLV infection when histopathology is compatible with HD and Non Hodgkin Lymphoma.

Aged↗
PubMed1992

Molecular analysis of the Bacillus subtilis bacteriophage SPP1 region encompassing genes 1 to 6. The products of gene 1 and gene 2 are required for pac cleavage.

Packaging of Bacillus subtilis phage SPP1 DNA into viral capsids is initiated at a specific DNA site termed pac. Using an in vivo assay for pac cleavage, we show that initiation of DNA synthesis and DNA packaging are uncoupled. When the DNA products of pac cleavage were analyzed, we could detect the pac end that was destined to be packaged, but we failed to detect the other end of the cleavage reaction. SPP1 conditional lethal mutants, which map adjacent to pac, were analyzed with our assay. This revealed that the products of gene 1 and gene 2 are essential for pac cleavage. SPP1 mutants that are affected in the genes necessary for viral capsid formation (gene 41) or involved in headful cleavage (gene 6) remain proficient in pac site cleavage. Analysis of the nucleotide sequence (2.769 x 10(3) base-pairs) of the region of the genes required for pac cleavage revealed five presumptive genes. We have assigned gene 1 and gene 2 to two of these open reading frames (orf), giving the gene order gene 1-gene 2-orf 3-orf 4-orf 5. The direction of transcription of the gene 1 to orf 5 operon and the length of the mRNAs was determined. We have identified, upstream from gene 1, the major transcriptional start point (P1). Transcription originating from P1 requires a phage-encoded factor for activity. The organization of gene 1 and gene 2 of SPP1 resembles the organization of genes in the pac/cos region of different Escherichia coli double-stranded DNA phages. We propose that the conserved gene organization is representative of the packaging machinery of a primordial packaging system.

Amino Acid Sequence↗
PubMed1992

The delta-endotoxin protein family displays a hydrophobic motif that might be implicated in toxicity.

A computer-based analysis of hydropathy and surface probability of representative members of each class of the Cry family of proteins was performed. A highly conserved hydrophobic motif within the previously described block, D2, is present not only in lepidopteran toxin genes but also in toxins active against diptera and coleoptera. An interesting feature of this hydrophobic motif is the presence of an aspartic residue (highly hydrophilic) in its middle part. Comparison with the amino acid sequence from diphtheria toxin showed that it also contains a hydrophobic motif similar to the one present in the Bacillus thuringiensis toxins. It also contains an aspartic residue in the middle part and some speculations are presented on the function of this specific region with regard to the toxic mechanism of action.

Amino Acid Sequence↗
PubMed1991

Recombination-dependent concatemeric plasmid replication.

The replication of covalently closed circular supercoiled (form I) DNA in prokaryotes is generally controlled at the initiation level by a rate-limiting effector. Once initiated, replication proceeds via one of two possible modes (theta or sigma replication) which do not rely on functions involved in DNA repair and general recombination. Recently, a novel plasmid replication mode, leading to the accumulation of linear multigenome-length plasmid concatemers in both gram-positive and gram-negative bacteria, has been described. Unlike form I DNA replication, an intermediate recombination step is most probably involved in the initiation of concatemeric plasmid DNA replication. On the basis of structural and functional studies, we infer that recombination-dependent plasmid replication shares important features with phage late replication modes and, in several aspects, parallels the synthesis of plasmid concatemers in phage-infected cells. The characterization of the concatemeric plasmid replication mode has allowed new insights into the mechanisms of DNA replication and recombination in prokaryotes.

Bacteria↗
PubMed1990

The generation of concatemeric plasmid DNA in Bacillus subtilis as a consequence of bacteriophage SPP1 infection.

Bacteriophage SPP1 infection of Bacillus subtilis cells bearing plasmids induces the synthesis of multigenome-length plasmid molecules. Two independent pathways can account for this synthesis. In one of those, homology to the phage genome is required, whereas in the other such homology is not a prerequisite. In wild type cells both modes overlap. In dnaB(Ts), at non permissive temperature, or in recE polA strains the main concatemeric plasmid replication mode is the homology-dependent plasmid (hdp) mode. The rate of recombination-dependent concatemeric plasmid DNA synthesis is a consequence of a phage-plasmid interaction which leads to chimeric phage::plasmid DNA. The second mode, which is an homology-independent plasmid (hip) mode seems to be triggered upon the synthesis of a phage encoded product(s) (e.g. inactivation of the exonuclease V enzyme).

Bacillus subtilis↗
PubMed1990

Functional analysis of the Bacillus subtilis bacteriophage SPP1 pac site.

Encapsidation of the DNA of the virulent Bacillus subtilis phage SPP1 follows a processive unidirectional headful-mechanism and initiates at a unique genomic location (pac). We have cloned a fragment of SPP1 DNA containing the pac site flanked by reporter genes into the chromosome of B. subtilis. Infection of such cells with SPP1 led to highly efficient packaging, initiated at the inserted pac site, of chromosomal DNA. The directionality in the packaging of this DNA was the same as observed with vegetative phage DNA. Mutagenizing the chromosomal pac insert defined an 83 base pair segment containing the pac cleavage site which is sufficient to direct phage specific DNA encapsidation. The packaging recognition signal as defined can also be utilized by the SPP1 related phages 41c, SF6 and rho 15.

Bacillus subtilis↗
PubMed1989

Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity.

A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.

Crosses, Genetic↗
PubMed1988

Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1.

The proteins P10 and P12 have been shown to be gene products of a new stability system, ParD, of plasmid R1. It is now shown that an R1 miniplasmid, pAB112, carrying a trans-complementable amber mutation in the gene of the P10 protein, is lethal for the host in the absence of suppression. This lethal effect is suppressed in a supF background and also by deletions in pAB112 that affect the gene of the P12 protein. These data indicate that the P12 protein has a lethal effect on the host and that this effect is neutralized by the P10 protein. The possibility that the stabilization conferred by the ParD system could be due to a counterselection, mediated by P12, of cells that lose the plasmid at cell division, is discussed.

Escherichia coli↗
PubMed1988

Ammonium assimilation in Rhizobium phaseoli by the glutamine synthetase-glutamate synthase pathway.

Evidence from in vitro and in vivo studies showed that in Rhizobium phaseoli ammonium is assimilated by the glutamine synthetase (GS)-glutamate synthase NADPH pathway. No glutamate dehydrogenase activity was detected. R. phaseoli has two GS enzymes, as do other rhizobia. The two GS activities are regulated on the basis of the requirement for low (GSI) or high (GSII) ammonium assimilation. When the 2-oxoglutarate/glutamine ratio decreases, GSI is adenylylated. When GSI is inactivated, GSII is induced. However, induction of GSII activity varied depending on the rate of change of this ratio. GSII was inactivated after the addition of high ammonium concentrations, when the 2-oxoglutarate/glutamine ratio decreased rapidly. Ammonium inactivation resulted in alteration of the catalytic and physical properties of GSII. GSII inactivation was not relieved by shifting of the cultures to glutamate. After GSII inactivation, ammonium was excreted into the medium. Glutamate synthase activity was inhibited by some organic acids and repressed when cells were grown with glutamate as the nitrogen source.

Culture Media↗
PubMed1988

Introduction of the Escherichia coli gdhA gene into Rhizobium phaseoli: effect on nitrogen fixation.

Rhizobium phaseoli lacks glutamate dehydrogenase (GDH) and assimilates ammonium by the glutamine synthetase-glutamate synthase pathway. A strain of R. phaseoli harboring the Escherichia coli GDH structural gene (gdhA) was constructed. GDH activity was expressed in R. phaseoli in the free-living state and in symbiosis. Nodules with bacteroids that expressed GDH activity had severe impairment of nitrogen fixation. Also, R. phaseoli cells that lost GDH activity and assimilated ammonium by the glutamine synthetase-glutamate synthase pathway preferentially nodulated Phaseolus vulgaris.

Cloning, Molecular↗
PubMed1987

Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid.

We provide evidence that a mutation which derepresses an autoregulated system that is located in the vicinity of the basic replicon of R1, stabilizes the ParA- and ParB- miniplasmid of R1 pKN1562, without increasing its copy number. The system, which we have called ParD, maps inside the 1.45-kb PstI-EcoRI fragment that is adjacent to the origin of replication of the plasmid. Two proteins whose expression is coordinated are components of the system. The sequence of the PstI-EcoRI fragment was obtained. The wild-type ParD system determines in cis a basal but detectable stability.

Amino Acid Sequence↗