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P Avila

Publications and source records attributed to P Avila.

At least 19 recordsLinked to original sources
PubMed2000

Two active-site tyrosyl residues of protein TrwC act sequentially at the origin of transfer during plasmid R388 conjugation.

Protein TrwC is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation. Site-directed mutagenesis was used to change to phenylalanine each of a set of four conserved tyrosyl residues in the sequence of the N-terminal relaxation domain of the protein. Simultaneous mutation of both Y18 and Y26 was required to abolish in vitro cleavage and strand-transfer reactions catalyzed by protein TrwC on oligonucleotides containing the nic site. Thus, both Y18 and Y26 could be involved independently in the formation of oligonucleotide-protein covalent complexes that constitute presumed intermediates of these reactions. This hypothesis was confirmed by the observation of Y18 and Y26-specific peptide-oligonucleotide adducts after protease digestion of TrwC and mutant derivatives. Finally mutation Y18F, but not mutation Y26F, abolished nic-cleavage of a supercoiled DNA containing the R388 origin of transfer (oriT). These data allowed the construction of a model for conjugative DNA processing in which Y18 specifically catalyzes the initial cleavage reaction, while Y26 is used for the second strand-transfer reaction, which terminates conjugation. The model suggests a control mechanism that can be effective at each conjugative replication cycle.

Amino Acid Sequence↗
PubMed1997

Genes involved in conjugative DNA processing of plasmid R6K.

The conjugative transfer region of the IncX plasmid R6K (TRA(x)) was analysed by transposon mutagenesis and DNA sequencing. Tn5tac1 insertional mutations localized TRA(x) to a 14.8 kb segment containing the alpha origin of transfer (oriT alpha), genes involved in conjugative DNA-processing (Dtr(x)) and genes involved in pilus synthesis and assembly (Mpf(x)). A second functional oriT, oriTbeta, was located at a distance of 5.3 kb from oriT alpha and was outside TRA(x). Mpf(x) occupied a segment of 10kb, as judged by the location of insertions conferring resistance to infection by the X pilus-specific phage X-2. At both sides of Mpf(x) there were insertions that were Tra but X-2 sensitive, suggesting that the mutations were in Dtr(x). This region was sequenced and three genes were identified: taxA, taxB, and taxC. The overall organization was oriT alpha-taxA-taxC-Mpf(x)-taxB. taxC coded for a oriT-relaxase that belongs to the VirD2 family. taxA coded for a protein of 181 amino acids that showed similarity to TraY of F-like plasmids and to the Arc-repressor superfamily. TaxB showed similarity to TraG-like proteins, a protein superfamily probably involved in coupling the relaxosome to the DNA-transport apparatus. TaxA and TaxC are required for oriT nicking in vivo. The nicking reaction was mistakenly assumed by Flashner et al. (1996) to represent a feature of the vegetative replication origins. However, insertions or deletions disrupting taxA and taxC affected conjugation but not replication of R6K. Conversely, protein pi, which is absolutely required for replication of R6K, was not required for conjugative transfer. In addition, protein DDP3, which is also assumed to have a role in replication, was found to be a positive modulator of bacterial conjugation. Taken together, these results rule out a direct and essential involvement of conjugation proteins in R6K vegetative replication, and also rule out the requirement of replication protein pi for conjugation.

Amino Acid Sequence↗
PubMed1997

A hot spot in plasmid F for site-specific recombination mediated by Tn21 integron integrase.

Integron In2 integrase (IntI1)-mediated site-specific recombination between two primary sites occurs at a high frequency, while that between a primary and a secondary site occurs at frequencies around 10,000 times lower. Secondary sites consist of a pentanucleotide with only two fully conserved residues (GWTMW). The analysis of IntI1-mediated recombinants in the plasmid pOX38 revealed the existence in this plasmid of a site used at a frequency intermediate between those of primary and secondary sites. Analysis of this site showed two potentially relevant structural features: first, a set of two consensus pentanucleotides, separated by 5 bp and in opposite orientations, forming what will be called a double site; and second, a longer sequence with some extent of sequence symmetry with the double site at its 3' end. A recombinant plasmid, pSU18P, containing a double site was constructed. Examination of R388-pSU18P recombinants showed that double sites were used preferentially over single pentanucleotides by IntI1. Comparisons of the nucleotide sequences of known 59-bp elements showed that in most cases there was a double site at each element end. Mutagenesis of the F hot spot was carried out to make it look more like the consensus 59-bp element. The improved sites showed recombination frequencies and specificities almost comparable to those observed at IntI1 primary sites.

Base Sequence↗
PubMed1996

Plasmid R6K contains two functional oriTs which can assemble simultaneously in relaxosomes in vivo.

Plasmid R6K contains two functional origins or transfer (oriT), in contrast to previously characterized conjugative plasmids. The oriTs are formed by 98 bp palindromic sequences invertedly orientated with respect to each other and located in the immediate vicinity of the alpha and beta origins of replication. The gene for R6K oriT-nickase, taxC, was identified by transposon mutagenesis and sequenced, revealing that TaxC belongs to the VirD2 nickase family. The protein was overproduced and purified. It catalysed a cleaving-joining reaction on single-stranded DNA containing its target sequence. Identification of the nic sites suggested that the R6K oriTs belong to the RP4/VirD2 oriT family. Cleavage was highly specific and did not occur with oligonucleotides cleaved by related nickases like TraI of RP4 or VirD2 of the Ti plasmid. nic cleavage of in vivo preassembled relaxation complexes was induced by incubation of plasmid cleared lysates with ethidium bromide. Nicked molecules obtained in this way were treated with snake venom phosphodiesterase to produce double strand cleavages at the nic sites. 35% of the molecules were cleaved simultaneously at both nic sites, both in the case of R6K and of R6Kdrd1, a derepressed mutant whose frequency of transfer is 1000-fold higher. This figure represents the minimum percentage of individual R6K molecules containing two pre-assembled relaxation complexes.

Amino Acid Sequence↗
PubMed1994

Physical activity training for weight loss in Latinas: a controlled trial.

This study tested the effects of an exercise and diet modification training program for weight loss among Latinas. Forty four obese women were assigned to an experimental training group (n = 22) or a control (n = 22), at random. One session per week for eight weeks included instruction for diet modification and walking for exercise, and all women were led in 20 min of walking during each session. Instruction was provided by a bi-cultural Spanish-speaking physician. Statistically significant (P < 0.05) decreases, relative to controls, were obtained for Body Mass Index, waist to hip ratio, and serum cholesterol. Significant increases were obtained for fitness, exercise rate and frequency, and diet/exercise knowledge. Results suggest that the training was effective for decreasing obesity and increasing fitness among Low SES, Mexican-American women. Implications for weight control and disease prevention among under-served populations are discussed.

Adult↗
PubMed1993

[Alcohol drinking in parents and its relation with intellectual score of their children].

Among children living in orphanages of the Metropolitan Region, the paternal alcohol ingestion patterns were investigated. According to abnormal parental alcohol ingestion, a sample of 291 children was studied, of whom 32% were living in simple protection homes for infants with normal intellectual coefficient and 52% in homes for mentally retarded boys. There was a higher frequency and intensity of abnormal alcohol ingestion among parents of infants living in homes for the mentally retarded. Among these, 29% of mothers were illiterate and 11% had incomplete primary school education compared to mothers of simple protection homes in whom the frequency of illiteracy was 10% and of incomplete primary education, 50%. Twenty percent of fathers of children living in homes for the mentally retarded were unemployed, 16% had occasional jobs and 11% had a stable work; these numbers were 11, 30 and 15% respectively for fathers of simple protection homes. Among infants living in homes for the mentally retarded, undernutrition was found in 44%, growth retardation in 51% and microcephaly in 34% compared to 33, 30 and 11% in children living in simple protection homes. Sixteen percent of the sample had fetal alcohol syndrome, 24% among those living in homes for the mentally retarded and 12% in simple protection homes. There was an inverse relationship between offspring intellectual coefficient and the number of drinking parents.

Adolescent↗
PubMed1991

[Cough caused by angiotensin-converting enzyme inhibitors. A series of cases collected by spontaneous notification of adverse reactions].

Cough is one of the possible untoward adverse drug effects of angiotensin converting enzyme inhibitors. We describe the available information on 50 cough episodes attributable to captopril and 18 episodes attributable to enalapril reported to the Spanish Drug Surveillance System. Cough represented 37% and 39% of the reports of side effects of captopril and enalapril, respectively. There was a remarkable female predominance among the patients with cough. Cough developed at very low doses (15 mg of captopril and 5 mg of enalapril daily), although the patients on captopril who developed cough were receiving higher doses than those who presented other side effects. A high proportion of patients (29%) continued with the drug for more than six months after cough had developed, suggesting the need for a wider knowledge of this side effect.

Aged↗
PubMed1990

Site-specific recombination by mutants of Tn21 resolvase with DNA recognition functions from Tn3 resolvase.

The resolvases from the transposons Tn3 and Tn21 are homologous proteins but they possess distinct specificities for the DNA sequence at their respective res sites. The DNA binding domain of resolvase contains an amino acid sequence that can be aligned with the helix-turn-helix motif of other DNA binding proteins. Mutations in the gene for Tn21 resolvase were made by replacing the section of DNA that codes for the helix-turn-helix with synthetic oligonucleotides. Each mutation substituted one amino acid in Tn21 resolvase with either the corresponding residue from Tn3 resolvase or a residue that lacks hydrogen bonding functions. The ability of these proteins to mediate recombination between res sites from either Tn21 or Tn3 was measured in vivo and in vitro. With one exception, where a glutamate residue had been replaced by leucine, the activity of these mutants was similar to that of wild-type Tn21 resolvase. A further mutation was made in which the complete recognition helix of Tn21 resolvase was replaced with that from Tn3 resolvase. This protein retained activity in recombining Tn21 res sites, though at a reduced level relative to wild-type; the reduction can be assigned entirely to weakened binding to this DNA. Neither this mutant nor any other derivative of Tn21 resolvase had any detectable activity for recombination between res sites from Tn3. The exchange of this section of amino acid sequence between the two resolvases is therefore insufficient to alter the DNA sequence specificity for recombination.

Amino Acid Sequence↗
PubMed1990

DNA binding by mutants of Tn21 resolvase with DNA recognition functions from Tn3 resolvase.

Substitution of amino acids within the section of Tn21 resolvase that corresponds to a helix-turn-helix structure, with the equivalent residues from Tn3 resolvase, yields proteins that retain the ability to mediate recombination between res sites from Tn21. These proteins had no recombinational activity on res sites from Tn3, even when the complete recognition helix had been exchanged. In this study, the binding of these mutants of Tn21 resolvase to DNA fragments containing res from either Tn21 or Tn3 was analysed by DNase I footprinting and by gel retardation. With DNA containing res from Tn21, the mutants bound to all three of the binding sites for resolvase (I, II, and III) but with a lower affinity than wild-type Tn21 resolvase. No complexes were detected between Tn3 resolvase and Tn21 DNA. With DNA containing res from Tn3, both the mutants and wild-type Tn21 resolvase bound to sites II and III, forming similar complexes to those with Tn3 resolvase: some of the mutants had higher affinities for these two sites on Tn3 DNA than on Tn21 DNA. In contrast, at site I in res from Tn3 (the location of the recombinational cross-over), the derivatives of Tn21 resolvase formed aberrant complexes whose structures differed radically from that with Tn3 resolvase. Alterations in the amino acid sequence of resolvase, within the helix-turn-helix region, therefore modulate the affinity of the protein for its target sequence in the DNA, but the specificity of resolvase for recombination at its cognate res sites is determined by the resultant organization of the DNA-protein complex.

Base Sequence↗
PubMed1990

General organization of the conjugal transfer genes of the IncW plasmid R388 and interactions between R388 and IncN and IncP plasmids.

The complete conjugal transfer gene region of the IncW plasmid R388 has been cloned in multicopy vector plasmids and mapped to a contiguous 14.9-kilobase segment by insertion mutagenesis. The fertility of the cloned region could still be inhibited by a coresident IncP plasmid. The transfer region has been dissected into two regions, one involved in pilus synthesis and assembly (PILW), and the other involved in conjugal DNA metabolism (MOBW). They have been separately cloned. PILW also contains the genes involved in entry exclusion. MOBW contains oriT and the gene products required for efficient mobilization by PILW. MOBW plasmids could also be mobilized efficiently by PILN, the specific pilus of the IncN plasmid pCU1, but not by PILP, the specific pilus of the IncP plasmid RP1.

Cloning, Molecular↗
PubMed1988

Analysis of the variable endpoints generated by one-ended transposition of Tn21.

One-ended transposition of Tn21 generates recombinants usually containing a whole copy of the donor replicon plus a short duplication of it (S. Mötsch, R. Schmitt, P. Avila, F. de la Crue, E. Ward, and J. Grinsted, Nucleic Acids Res. 13:3335-3342, 1985). This work shows that recombinants containing less than a whole copy of the donor replicon (hereafter called short recombinants) could also be detected when plasmid donors which contained two selectable genetic markers were used. Short recombinants were produced at the same frequency from TnpR+ donor molecules as from TnpR- donor molecules in a RecA- background. Therefore, they were not resolution products of larger recombinants. This result invalidates a previous hypothesis to explain one-ended transposition, that is, that one-ended transposition arises from the use of secondary ends by the transposition apparatus. On the other hand, it suggests that one-ended transposition of Tn21 occurs via a simple insertion mechanism.

Base Sequence↗
PubMed1985

Junction sequences generated by 'one-ended transposition'.

In the presence of the cognate transposase, plasmids containing a single inverted repeat (IR) sequence of Tn21 or of Tn1721 can fuse efficiently with other plasmids ('one-ended transposition'). The junctions across the sequences of donor and recipient DNA in recombinants generated by this process have been determined. These show that the segment of donor DNA starts precisely at the IR sequence (it is variable at the other end), and is flanked by a direct repeat of host DNA (usually 5bp) that was present only once in the original host sequence. These are characteristics of recombinants generated by transposition of Tn21 and Tn1721 themselves, suggesting that the mechanism of one-ended transposition is very similar to that of the corresponding entire elements.

Base Sequence↗
PubMed1984

Plasmids containing one inverted repeat of Tn21 can fuse with other plasmids in the presence of Tn21 transposase.

In the presence of the Tn21 transposase, plasmids that contain a single Tn21 inverted repeat sequence fuse efficiently with other plasmids. This reaction occurs in recA strains, is independent of the transposon-encoded resolution system, and results in insertions into different sites in the recipient plasmid. All fusion products studied contained at least one complete copy of the donor plasmid; most also contained some duplication of it as well. The data are consistent with processive models of transposition.

Base Sequence↗