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Discurso leido el 25 de marzo de 1994.

Publicado por dicha Capitanía en 1984.

Disertación leída en la Sesión del día 19/1/87

Populations of RNA and ssDNA viruses within their hosts contain a heterogeneous collection of variant genomes known as quasispecies. Large variability in mitochondrial DNA has also been found within the same organism, drawing an interesting parallel between the two situations. The advent of next-generation sequencing technologies facilitated studying genetic variation, but many open-source bioinformatic tools have to be combined in a non-trivial approach. Here it is presented QuasiFlow, a workflow based on well-stablished software that extracts reliable mutations and recombinations, even at low frequencies (~10-4), provided that at least 250 million nucleotides are analysed. Accurate prediction of mutations and recombinations has been demonstrated with synthetic reads and with in vitro rolling-circle amplification of a plant geminivirus. An in-depth analysis of viral quasispecies was performed and QuasiFlow revealed the coexistence in the plant of three virus genomes and distinct recombinations between some of them. Human mitochondrial variants were also investigated and high level of heteroplasmy (75%) was confirmed, and the relation between low-frequency heteroplasmy (0.1- 0.2%) and some human diseases, regardless of sex, was established. Hence, we propose that QuasiFlow may find use with known and emerging viruses to reveal evolutionary jumps and co-infections, with mitochondrial DNA to detect relevant heteroplasmy would otherwise be elusive, or even in other population studies such as those considering single cell sequencing., This work was supported by Consejería de Economía, Innovación y Ciencia, Junta de Andalucía with assistance from the European Regional Development Fund (ERDF) and the European Social Fund (ESF) [grant to Research Groups BIO-264 and BIO-267; P10594 -CVI- 6561 and UMA18-FEDERJA178 to A.G.-P., E.V.M; UMA20-FEDERJA-029 to MGC; and a predoctoral fellowship to L.D.-M. A.G-P. acknowledges support of Plan Propio de Investigación y Transferencia of the University of Málaga. Open access charges was funded by University of Málaga. The authors also thankfully acknowledge the computer resources and the technical support provided by the Plataforma Andaluza de Bioinformática of the University of Málaga.

9 p.-6 fig., We present a computer model to predict the patterns expected for the replication intermediates (RIs) of DNA fragments analyzed by neutral/neutral two-dimensional (2D) agarose gel electrophoresis. The model relies on the mode of replication (uni- or bi-directional), the electrophoretic mobility of linear DNA fragments and the retardation caused by the three-dimensional shape of non-linear molecules. The utility of this model is demonstrated with two examples: replication analysis of the plasmids pBR322 and pHH5.8 in Escherichia coli after digestions with EcoRI and HindIII, respectively., This work was partially supported by Grant 96/0470 from the Spanish Fondo de Investigación Sanitaria,Grant PM95/0016 from the Spanish Dirección General de Enseñanza Superior,Grant 08.6/0016/1997 from the Comunidad Autónoma de Madrid and Grant TIC96-1125-C03-01 from the Spanish Comisión Interministerial de Ciencia y Tecnología (CICYT ).

9 p.-8 fig., Co-orientation of replication origins is the most common organization found in nature for multimeric plasmids. Streptococcus pyogenes broad-host-range plasmid pSM19035 and Escherichia coli pPI21 are among the exceptions. pPI21, which is a derivative of pSM19035 and pBR322, has two long inverted repeats, each one containing a potentially active ColE1 unidirectional origin. Analysis of pPI21 replication intermediates (RIs) by two-dimensional agarose gel electrophoresis and electron microscopy revealed the accumulation of a specific RI containing a single internal bubble. The data obtained demonstrated that initiation of DNA replication occurred at a single origin in pPI21. Progression of the replicating fork initiated at either of the two potential origins was transiently stalled at the other inversely oriented silent ColE1 origin of the plasmid. The accumulated RIs, containing an internal bubble, occurred as a series of stereoisomers with different numbers of knots in their replicated portion. These observations provide one of the first functional explanations for the disadvantage of head-to-head plasmid multimers with respect to head-to-tail ones., This work was partially supported by Grants 93/0161 and 96/0470 from the Spanish Fondo de Investigación Sanitaria and Grant AE00118/94 from the Comunidad Autónoma de Madrid, Madrid, Spain.