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E Domingo

Publications and source records attributed to E Domingo.

At least 235 records · Page 13Linked to original sources
PubMed1980

Nucleotide sequence heterogeneity of the RNA from a natural population of foot-and-mouth-disease virus.

The genomic RNA from isolates of foot-and-mouth-disease virus (FMDV) of serological types O or C obtained during epizootic outbreaks have been analysed by two-dimensional gel electrophoresis of the T1 RNase-generated oligonucleotides (T1 fingerprinting). Among virus isolates that are closely related serologically, 4-12 oligonucleotide changes were detected constitute the genome, the variations affect 0.7%-2.2% positions in FMDV RNA. Higher nucleotide-sequence divergence exists between the genomic RNAs from serologically unrelated viruses, while a 100-fold lower RNA sequence heterogeneity has been detected by analysis of individual clones derived from one viral isolate. Oligonucleotide mapping indicates that the variant oligonucleotides are scattered throughout the FMDV genome. We suggest that extensive genetic variability at many RNA sites is the basis for the antigenic diversity of FMDV.

Antigens, Viral↗
PubMed1978

Nucleotide sequence heterogeneity of an RNA phage population.

The nucleotide sequence of 32P-RNA from Q beta phage clones was sampled by two-dimensional polyacrylamide gel electrophoresis of the RNAase T1-resistant oligonucleotides (T1 fingerprinting). About 15% of the clones derived from a multiply passaged Q beta population showed fingerprint patterns which deviated from that of the RNA from the total population. All deviations examined could be attributed to one and, less frequently, to two or more nucleotide transitions. Since the fingerprinting technique allows the analysis of only about 10% of the RNA sequence, we estimate that each viable phage genome in a multiply passaged population differs in one to two positions from the "average" sequence of the parental population. Several deviant clones were tested by growth competition against a "wildtype" population, after 10-20 generations, the resulting phage showed the "wild-type" T1 fingerprint pattern. We propose that a Q beta phage population is in a dynamic equilibrium, with viable mutants arising at a high rate (Batschelet, Domingo and Weissmann, 1976; Domingo, Flavell and Weissmann, 1976) on the one hand, and being strongly selected against on the other. The genome of Q beta phage cannot be described as a defined unique structure, but rather as a weighted average of a large number of different individual sequences.

Base Sequence↗
PubMed1976

In vitro site-directed mutagenesis: generation and properties of an infectious extracistronic mutant of bacteriophage Qbeta.

An infectious extracistronic mutant of phage Qbeta has been prepared by site-directed mutagenesis. Qbeta RNA minus strands containing the mutagenic base analog N4-hydroxy-CMP instead of UMP at position 39 from the 5' end were synthesized in vitro and used as template for Qbeta replicase to synthesize one generation of plus strands. E. coli spheroplasts were infected with the newly synthesized plus strands and phage recovered from single plaques. RNA sequence analysis revealed that four out of the eighteen phage clones analyzed contained RNA with an A leads to G transition at position 40 from the 3' end (which corresponds to position 39 of the minus strand). Thus, the viability of phage Qbeta does not depend on a unique nucleotide sequence in the 3'-extracistronic RNA segment. Upon in vivo propagation of mutant 40, spontaneous true revertants arose with high frequency and overgrew the parental clone within about 10 passages, indicating a selective disadvantage of the extracistronic mutant. Replication of mixtures of wild type and mutant RNA in vitro resulted in a decrease of the proportion of mutated RNA in the progeny plus strands. The fact that Qbeta RNA containing an A leads to G transition in nucleotide--40 of Qbeta RNA is less efficiently replicated in vitro may explain the selective disadvantage of the mutant phage in vivo. The preparation of an infectious mutated RNA by site-directed mutagenesis shows that the method is suitable to produce specific nucleotide exchanges without impairing the biological competence of the RNA.

Bacteriophages↗
PubMed1975

Temperature and salt effects on the formation of preinitiation complexes between RNA polymerase and phage DNA.

The influence of temperature and KCl concentration on the formation of rifampicin-resistant preinitiation complexes by holo RNA polymerase has been compared for T4 DNA and Azotobacter phage A21 DNA. The sharp transition with respect to temperature between an inactive complex of polymerase and DNA and a preinitiation complex reflects an equilibrium between the two complexes, the position of which depends on the temperature and the salt concentration. The transition is shifted to higher temperatures by increasing the KCl concentration. The position of this transition is characteristically different for T4 and A21 DNA. The midpoint for A21 DNA is about 15 degrees C above that for T4 at 0.006 M KCl. At 0.15 M KCl the transition for A21 DNA cannot be observed below 37 degrees C. This difference is responsible for the apparent inhibition of a21 dna transcription by KCl and for the low template activity of A21 DNA under the conditions of the standard assay. Both holo and core RNA polymerases are able to form complexes with A21 DNA that are resistant to attack by rifampicin. The second-order rate constant for the inactivation of the complex with the core enxyme is three times greater than that for the complex with the holoenzyme.

Azotobacter↗
PubMed1975

Transcription of bacteriophage deoxyribonucleic acid. Comparison of Escherichia coli and Azotobacter vinelandii sigma subunits.

The effect of the sigma subunit of RNA polymerase on the rate and asymmetry of the in vitro transcription of Escherichia coli and Azotobacter vinelandii phage DNAs has been studied with purified E. coli and A. vinelandii RNA polymerases and hybrid enzymes containing the core subunits of one enzyme and sigma from the other. The effect of sigma on the rate of transcription is characteristic of the template and not of the enzyme and depends on ionic strength. The rate of transcription of A. vinelandii phage A21 DNA is decreased by sigma at high ionic strength, but shows the more characteristic stimulation at KCl concentrations below 0.05 M. In contrast, the stimulation by sigma of T4 DNA transcription increased with an increase in the KCl concentrations. All combinations of core and sigma subunits behaved similarly with respect to stimulation or inhibition by sigma and with respect to asymmetric transcription of S13 replicative form (RF)DNA. However, the heterologous, but not the homologous combinations of core and sigma transcribed A21 symmetrically. S13 RF DNA in the superhelical, but not in the relaxed configuration, is transcribed asymmetrically by the A. vinelandii core enzyme. A role for the core subunits in specific site recognition is indicated by this observation.

Animals↗
PubMed1975

Transcription of Azotobacter phage deoxyribonucleic acid. Salt-dependent equilibrium between steps in initiation.

The transcription of Azotobacter phage A21 DNA by Escherichia coli or Azotobacter vinelandii RNA polymerase differs from that of some other DNAs in its inhibition by moderate concentrations of KCl. This characteristic results in an apparent low template activity for this DNA as compared with T4 DNA under standard assay conditions. From an analysis of the dependence of the various steps in initiation on KCl it is concluded that the effect is exerted on an equilibrium between an inactive polymerase-DNA complex and an active preintitiation complex. This salt-sensitive equilibrium favors the inactive complex at a lower KCl concentration than with other templates. It can be approached from other low or high salt concentrations at a measurably slow rate.

Azotobacter↗
PubMedDate not supplied

Native-like cyclic peptide models of a viral antigenic site: finding a balance between rigidity and flexibility.

Antigenic site A of foot-and-mouth disease virus (serotype C) has been reproduced by means of cyclic versions of peptide A15, YTASARGDLAHLTTT, corresponding to residues 136-150 of envelope protein VP1. A structural basis for the design of the cyclic peptides is provided by crystallographic data from complexes between the Fab fragments of anti-site A monoclonal antibodies and A15, in which the bound peptide is folded into a quasi-cyclic pattern. Head-to-tail cyclizations of A15 do not provide peptides of superior antigenicity. Internal disulfide cyclization, however, leads to analogs which are recognized as one to two orders of magnitude better than linear A15 in both ELISA and biosensor experiments. CD and NMR studies show that the best antigen, CTASARGDLAHLTT-Ahx-C (disulfide), is very insensitive to environment-induced conformational change, suggesting that cyclization helps to stabilize a bioactive-like structure.

Amino Acid Sequence↗
PubMedDate not supplied

Postextrasystolic potentiation of systolic gradient in valvular aortic stenosis: clinical usefulness and analysis of hemodynamic factors.

To analyze the behavior of aortic valve gradient (AVG) after ventricular extrasystole (VE), we studied 36 pure valvular aortic stenoses (AS) free of coronary artery disease and obstructive hypertrophic cardiomyopathy, in whom basal (B) (74 +/- 32 mm Hg) and catheter-induced post-VE (110 +/- 50 mm Hg) AVG were obtained. In all 26 cases with valve area less than 0.70 cm2, the post-VE AVG was greater than or equal to 70 mm Hg. In 19 cases AVG after two or more consecutive VE were also obtained. Maximal post-VE AVG was obtained after multiple VE (19 cases) and/or after one VE causing a post-VE pause equal or longer than 1.7 basal cardiac cycles (post-VE RR greater than or equal to 1.7 B RR) (9 cases). Basal and postsingle VE AVG, up to a post-VE RR greater than or equal to 1.7 B RR, were a linear function of previous RR (r greater than or equal to 0.90), regression line slope increasing with AS severity (P = .05). Inotropic state measured by PEP/LVET only increased after multiple VE, P less than .01. AVG after multiple VE was independent of post-VE RR. Thus, 1) post-VE potentiation of AVG may be seen with fixed valvular AS without obstructive cardiomyopathy; 2) post-VE AVG is a function of compensatory pause after single VE and of increased inotropism after multiple VE; 3) analysis of maximal post-VE AVG generated as described is reliable and useful for assessing AS severity (post-VE AVG greater than or equal to 70 mm Hg meaning an aortic valve area less than 0.70 cm2) and may supplement valve area calculations.

Aortic Valve↗
PubMedDate not supplied

Synthetic peptides as functional mimics of a viral discontinuous antigenic site.

Functional reproduction of discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate into a single molecule each of the three protein loops that define the antigenic site. The site D mimics are designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulphide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194). Guinea pig antisera to the peptides recognize the viral particle and compete with site D-specific monoclonal antibodies, while inoculation with a simple (non-covalently bound) admixture of the three VP1-VP3 sequences yields no detectable virus-specific serum conversion. Similar results have been reproduced in two cattle. Antisera to the peptides are also moderately neutralizing of FMDV in cell culture and partially protective of guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent peptide-based vaccine against FMDV.

Amino Acid Sequence↗