Biomedical subjects

J Dopazo

Publications and source records attributed to J Dopazo.

At least 37 records · Page 2Linked to original sources

PubMed1997

A new index to find regions showing an unexpected variability or conservation in sequence alignments.

Present-day sequences are the result of long and complex pathways of evolution. Many of the features encoded in them are the result of a series of evolutionary processes, including selection, genetic drift, etc. Following this, protein families have a great amount of unexplored information that can be useful for many purposes beyond simple evolutionary studies. An index is presented here which permits the localization of regions showing an unexpected degree of variability or conservation. This index uses the information contained in sequence alignments and compares the observed over expected level of variability using tables for the relative likelihood of amino acid change.

Algorithms↗

PubMed1996

Point mutant frequencies in the pol gene of human immunodeficiency virus type 1 are two- to threefold lower than those of env.

Nucleotide sequences have been determined for the HIV-1 genomic regions encoding codons 41-108 and 181-219 of reverse transcriptase (RT) (pol gene), and codons 198-331 of gp120 (the C2V3 domains; env gene). Eighty-one HIV-1 samples from patients treated or untreated with RT inhibitors were used to sequence pol and 28 HIV-1 samples were used to sequence env. Several individual quasispecies have also been analyzed. All HIV-1 isolates belonged to subtype B. Point mutant frequencies and nucleotide diversities for pol were at most two- to threefold lower than env, with the proportion of nonsynonymous mutations ranging from 33 to 67%. A new variability index that takes into account the type of amino acid substitution as well as genetic distances between the compared sequences is introduced, and its main features for sequence comparisons emphasized. Extension of the calculations to gag, pol, and env sequences contained in the current HIV-1 database confirmed the high mutant frequencies for all HIV-1 genomic regions. The results indicate that although env is more tolerant to insertions and deletions than pol, point mutant frequencies for HIV-1 regions encoding nonstructural proteins are only two- to threefold lower than for regions encoding structural proteins. This implies the occurrence of frequent changes in HIV-1 phenotypes that are dependent on amino acid substitutions in viral enzymes.

Amino Acid Sequence↗

PubMed1996

A strain-type clustering of potato virus Y based on the genetic distance between isolates calculated by RFLP analysis of the amplified coat protein gene.

Potato virus Y (PVY) isolates have been classified into genetic strains by a host-independent criterion using a molecular typing method. The method used extracts from infected tissue, and included immunocapture-RT-PCR-RFLP analysis using 5 different restriction endonucleases (Dde I, Eco RV, Hinf I, Rsa I and Taq I). Genetic distances between the different PVY "restrictotypes" were calculated and used to define the PVY genetic strains. Three main clusters were found: PVYO, PVYN, and non-potato PVY (PVYNP), in good agreement with classical PVY strain definitions that combine different biological criteria. Our approach was incomparably quicker and more reliable and reproducible than biotyping. The potential of this approach for very quick, simple and automatable molecular epidemiological studies is discussed.

Antibodies, Monoclonal↗

PubMed1996

Phylogenetic relationships of european strains of porcine reproductive and respiratory syndrome virus (PRRSV) inferred from DNA sequences of putative ORF-5 and ORF-7 genes.

The complete ORF-5 gene and a fragment of the ORF-7 gene from 14 different European porcine reproductive and respiratory syndrome virus (PRRSV) isolates were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and their nucleotide sequences were determined. The ORF-7 gene displayed nucleotide and amino acid identities of 94.1-99.6% and 95.3-100% among isolates from different countries. The ORF-5 gene showed higher nucleotide (87.1-99.2% identity) and amino acid (-88% identity) variability. The resulting sequences were aligned with other European and North American PRRSV strains and phylogenetic relationships among these strains were established by the maximum parsimony method. The phylogenetic trees inferred from both genes were in agreement and showed that European and North American PRRSV strains clearly represent two different genotypes. According to both trees, there is a perfect correlation between strains and the countries in which they were isolated. Additionally, the phylogenetic position of European and North American PRRSV strains within the recently proposed family Arteriviridae was also analyzed.

Animals↗

PubMed1996

Assessing dissimilarity of genes by comparing their RNAse A mismatch cleavage patterns.

We propose a simple algorithm for estimating the number of nucleotide differences between a pair of RNA or DNA sequences through comparison of their RNAse A mismatch cleavage patterns. In the RNAse A mismatch cleavage technique two or more sample sequences are hybridized to the same RNA probe, the hybrids are partially digested with RNAse A, and the digestion products are compared on an electrophoretic gel. Here we provide an algorithm for converting the numbers of unique and matching electrophoretic bands into an estimate of the number of nucleotide differences between the sequences. Computer simulation indicates that the proposed method yields a robust estimate of the genetic distance despite stochastic errors and occasional violation of certain assumptions. Our study suggests that the method performs best when the distance between the sequences is < 15 differences. When the sequences under analysis are likely to have larger distances, we advise to substitute one long riboprobe with a set of shorter nonoverlapping probes. The new algorithm is applied to infer the proximity of several strains of pseudorabies virus.

Algorithms↗

PubMed1995

The CD36, CLA-1 (CD36L1), and LIMPII (CD36L2) gene family: cellular distribution, chromosomal location, and genetic evolution.

CD36, CLA-1, and LIMPII are single polypeptide membrane glycoproteins, and the genes encoding them constitute a recently described gene family (D. Calvo and M. A. Vega (1993) J. Biol. Chem. 268: 18929). In the present paper, a cDNA encoding the human lysosomal membrane protein LIMPII was used to determine its expression pattern in cells of various lineages. Like CLA-1, and in contrast with the restricted expression of CD36, the expression of LIMPII is widespread. Mapping of the human LIMPII and CLA-1 genes (gene symbols CD36L2 and CD36L1, respectively) to specific chromosomes revealed that CLA-1, LIMPII, and CD36 do not form a gene cluster, but are found dispersed on chromosomes 12, 4, and 7, respectively. These data, together with the phylogenetic analysis carried out for the members of this family, indicate that the LIMPII, CLA-1, and CD36 genes diverged early in evolution from an ancestor gene, possibly before the divergence between the arthropods and the vertebrates.

Amino Acid Sequence↗

PubMed1995

Evolutionary analysis of the picornavirus family.

An exhaustive evolutionary analysis of the picornavirus family has been carried out using the amino acid sequences of several proteins of the viruses including: the capsid proteins (1D, 1B, and 1C) situated at the 5' end of the genome and responsible for the serotype of the viruses, and the viral polymerase (3D), located at the 3' end of the genome. The evolutionary relationships found among the viruses studied support the new classification, recently suggested, in contrast to the classical one, and the existence of a new genus for the picornavirus family. In the new taxonomic organization, five genera form the picornavirus family: (1) aphthoviruses, (2) cardioviruses, (3) hepatoviruses (previously classified as enteroviruses), (4) renteroviruses (which mainly constitute a combination of the previous genera rhinovirus and enterovirus), and (5) a new genus, with a new and unique representative: the echovirus 22. Our analysis also allowed us, for the first time, to propose the most probable sequence of speciation events to have given rise to the current picornavirus family. The bootstrap procedure was used to check the reliability of the phylogenetic trees obtained. The application of the method of the statistical geometry in distance space to internal branches of the tree revealed a high degree of evolutionary "noise," which makes the resolution of some internal branching points difficult.

Capsid↗

PubMed1995

A method for determining the position and size of optimal sequence regions for phylogenetic analysis.

The availability of fast and accurate sequencing procedures along with the use of PCR has led to a proliferation of studies of variability at the molecular level in populations. Nevertheless, it is often impractical to examine long genomic stretches and a large number of individuals at the same time. In order to optimize this kind of study, we suggest a heuristic procedure for detection of the shortest region whose informational content can be considered sufficient for significant phylogenetic reconstruction. The method is based on the comparison of the pairwise genetic distances obtained from a set of sequences of reference to those obtained for different windows of variable size and position by means of a simple index. We also present an approach for testing whether the informative content in the stretches selected in this way is significantly different from the corresponding content shown by the larger genomic regions used as reference. Application of this test to the analysis of the VP1 protein gene of foot-and-mouth-disease type C virus allowed us to define optimal stretches whose informative content is not significantly different from that displayed by the complete VP1 sequence. We showed that the predictions made for type C sequences are valid for type O sequences, indicating that the results of the procedure are consistent.

Animals↗

PubMed1995

Sequence analysis of HIV-1 vif gene in Spanish isolates.

We have determined the nucleotide sequence of the HIV-1 vif gene in viruses obtained from symptomatic patients of distinct risk groups in Madrid. The genetic diversity among the isolates was estimated in 4.6% (+/- 1.4 standard deviation), a similar value to that obtained for the gag gene 3.9% (+/- 0.8 standard deviation) and env 4.1% (+/- 1 standard deviation) (Rojas et al., Virus Res 31, 331-342, 1994). Amino acid sequence analysis revealed the presence of hypermutable residues at positions 101 and 167, close to antigenically relevant sequential epitopes (comprising amino acids 87-94 and 172-178). Phylogenetic analysis supports the existence of two virus lineages circulating preferentially within different risk groups.

Adult↗

PubMed1995

Large deletions in the 5'-untranslated region of foot-and-mouth disease virus of serotype C.

Nucleotide sequences of the 5'-untranslated region (5'-UTR), at the 3'-side of the poly C tract, have been compared for 21 isolates of foot-and-mouth disease virus (FMDV) of serotype C from Europe, South America and The Philippines. A deletion of 43 nucleotides is present in the European isolates as compared with most American isolates. A larger deletion of 86 nucleotides is present in some viruses from South America and The Philippines. These deletions include the loss of one or two pseudoknot structures predicted in this region of the 5'-UTR. In addition, multiple point mutations have allowed the derivation of a phylogenetic tree which defines a grouping of isolates very similar to that derived from the capsid gene sequences of the same viruses. The study provides evidence that deletion (or addition) events must be very frequent during evolution of FMDV type C, since viruses which are phylogenetically very closely related (they belong to the same tree branch) may differ in the presence or absence of these deletions. Implications for FMDV evolution are discussed.

Animals↗

PubMed1995

Comparative study of the genetic variability in thymidine kinase and glycoprotein B genes of herpes simplex viruses by the RNase A mismatch cleavage method.

The variability of herpes simplex viruses has been measured using the RNAse A mismatch cleavage method in two genes: thymidine kinase and glycoprotein B of both HSV-1 and HSV-2. This technique permitted us to study the variability of the virus with a greater level of resolution than restriction endonuclease analysis. The phylogenetic trees obtained for the different genes allowed us to identify consistent clusters of viruses circulating in the same geographical area. Our results showed that thymidine kinase is more heterogeneous than glycoprotein B for both subtypes of HSV, and confirmed that HSV-1 is more heterogeneous than HSV-2 for both genes. This is the first time that this kind of analysis has been applied to DNA viruses.

Animals↗

PubMed1995

Primary genetic characterization of HIV-1 isolates from WHO-sponsored vaccine evaluation sites by the RNase-A mismatch method.

We have analyzed by the RNase-A mismatch method 35 isolates from four WHO-sponsored vaccine evaluation sites as a secondary laboratory of the WHO Network for HIV Isolation and Characterization. The application of an estimator for the establishment of genetic distances based on the RNase-A digestion patterns in combination with the phylogenetic analysis has allowed us to construct a tree with five well defined groups of viruses. Because the clustering with known reference strains, samples from Brazil could be grouped as subtype B and the majority of those from Thailand were subtype E. Some of the samples from Uganda were classified as subtype D. Isolates from Rwanda and some from Uganda were identified as subtype A viruses. These results coincide with data obtained by heteroduplex mobility assay and nucleotide sequencing in env regions. The RNase-A mismatch method combined with phylogenetic analysis permitted the primary genetic classification of 33 of 35 samples from the WHO Network.

AIDS Vaccines↗

PubMed1995

Molecular characterization of human immunodeficiency virus type 1 isolates from Venezuela.

Eight HIV-1 isolates from Venezuela have been characterized by nucleotide sequencing of the entire reverse transcriptase (RT)- and surface glycoprotein (gp 120)-coding regions. Average mutant frequencies were 2.5 x 10(-2) substitutions per nucleotide (s/nt) for the RT-coding region, and 10 x 10(-2) or 6.8 x 10(-2) s/nt for the gp120-coding region, depending on whether gaps introduced for optimal alignment were or were not, respectively, considered in the calculations. Phylogenetic trees were derived by maximum-likelihood, neighbor-joining, and maximum parsimony methods. In the trees derived from both RT- and gp120-coding regions, Venezuelan isolates cluster with subtype B viruses. However, the relative position of some of the isolates is considerably different in the two trees. Unique V3 loop amino acid sequences, not represented in the current database, have been identified among the Venezuelan isolates. In addition to representing the first molecular characterization of HIV-1 from Venezuela, the extensive genetic heterogeneity observed reinforces the interest in characterizing additional HIV-1 isolates worldwide for adequate vaccine design.

Adult↗

PubMed1995

An image-processing approach to dotplots: an X-Window-based program for interactive analysis of dotplots derived from sequence and structural data.

We present an approach to the study of the relationships between biological sequences and structures applying image analysis methods to dotplots. We introduce a set of analytical tools based on different types of digital image-processing filters that are new within the context of dotplots. We have reformulated some of the usual approaches in dotplot analysis as mathematical operations on images within the framework of mathematical morphology. An X-Window-based implementation of this new approach has been developed and is available by anonymous FTP.

Data Interpretation, Statistical↗

PubMed1994

Estimating errors and confidence intervals for branch lengths in phylogenetic trees by a bootstrap approach.

A method, based on the bootstrap procedure, is proposed for the estimation of branch-length errors and confidence intervals in a phylogenetic tree for which equal rates of substitution among lineages do not necessarily hold. The method can be used to test whether an estimated internodal distance is significantly greater than zero. In the application of the method, any estimator of genetic distances, as well as any tree reconstruction procedure (based on distance matrices), can be used. Also the method is not limited by the number of species involved in the phylogenetic tree. An example of the application of the method in the reconstruction of the phylogenetic tree for the four hominoid species--human, chimpanzee, gorilla, and orangutan--is shown.

Animals↗

PubMed1994

Monte Carlo simulation in phylogenies: an application to test the constancy of evolutionary rates.

Monte Carlo simulation has commonly been used in phylogenetic studies to test different tree-reconstruction methods, and consequently, its application for testing evolutionary models can be considered as a natural extension of this usage. Repetitive simulation of a given evolutionary process, under the restrictions imposed by the model to be tested, along a determinate tree topology allow the estimate of probability distributions for the desired parameters. Next, the phylogenetic tree can be reconstructed again without the constraints of the model, and the parameter of interest, derived from this tree, can be compared to the corresponding probability distribution derived from the restricted, simulated trees. As an example we have used Monte Carlo simulation to test the constancy of evolutionary rates in a set of cytochrome-c protein sequences.

Animals↗

PubMed1994

Immunogenicity of non-structural proteins of foot-and-mouth disease virus: differences between infected and vaccinated swine.

Non-structural as well as VP1 recombinant proteins of foot-and-mouth disease virus (FMDV) produced in E. coli, have been used to study the specific antibody response of infected or vaccinated swine. An analysis of sera from infected pigs, using a direct ELISA, showed that polypeptide 3ABC (spanning non-structural proteins 3A, 3B and 3C) was the most antigenic among the recombinant proteins studied and allowed specific detection of FMDV infected swine from the second week after the infection. The sensitivity of this assay was comparable to that obtained when the whole FMDV was used as ELISA antigen. Conversely, use of polypeptide 3ABC did not allow detection of significant levels of antibodies in sera from vaccinated animals. This differential pattern of ELISA reactivities offers a promising approach for the distinction of infected from vaccinated pigs. In addition, a highly specific and sensitive method of diagnosis for FMDV replication was achieved using an immunoblotting assay which detected antibodies against the 3ABC polypeptide.

Animals↗

PubMed1994

Molecular epidemiology of HIV-1 in Madrid.

Thirteen HIV-1 isolates from patients of different risk groups in Madrid (Spain) have been analyzed at the genetic level. Two distinct lineages of subtype B have been detected among the HIV-1 circulating in this area: one was related to SF-2/RF strains, whereas the other consists of a more heterogeneous group related to reference strain III-B. Variants of each lineage appeared to circulate preferentially within a risk group: III-B among intravenous drug users, and RF/SF-2 among male homosexuals.

Adult↗