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R Zardoya

Publications and source records attributed to R Zardoya.

32 records · Page 2Linked to original sources

Evolutionary relationships of the coelacanth, lungfishes, and tetrapods based on the 28S ribosomal RNA gene.

The origin of land vertebrates was one of the major transitions in the history of vertebrates. Yet, despite many studies that are based on either morphology or molecules, the phylogenetic relationships among tetrapods and the other two living groups of lobe-finned fishes, the coelacanth and the lungfishes, are still unresolved and debated. Knowledge of the relationships among these lineages, which originated back in the Devonian, has profound implications for the reconstruction of the evolutionary scenario of the conquest of land. We collected the largest molecular data set on this issue so far, about 3,500 base pairs from seven species of the large 28S nuclear ribosomal gene. All phylogenetic analyses (maximum parsimony, neighbor-joining, and maximum likelihood) point toward the hypothesis that lungfishes and coelacanths form a monophyletic group and are equally closely related to land vertebrates. This evolutionary hypothesis complicates the identification of morphological or physiological preadaptations that might have permitted the common ancestor of tetrapods to colonize land. This is because the reconstruction of its ancestral conditions would be hindered by the difficulty to separate uniquely derived characters from shared derived characters in the coelacanth/lungfish and tetrapod lineages. This molecular phylogeny aids in the reconstruction of morphological evolutionary steps by providing a framework; however, only paleontological evidence can determine the sequence of morphological acquisitions that allowed lobe-finned fishes to colonize land.

Animals↗

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↗

The complete nucleotide sequence of the mitochondrial genome of the lungfish (Protopterus dolloi) supports its phylogenetic position as a close relative of land vertebrates.

The complete DNA sequence (16,646 bp) of the mitochondrial genome of the African lungfish, Protopterus dolloi, was determined. The evolutionary position of lungfish as possibly the closest living relative among fish of land vertebrates made its mitochondrial DNA sequence particularly interesting. Its mitochondrial gene order conforms to the consensus vertebrate gene order. Several sequence motifs and secondary structures likely involved in the regulation of the initiation of replication and transcription of the mitochondrial genome are conserved in the lungfish and are more similar to those of land vertebrates than those of ray-finned fish. A novel feature discovered is that the putative origin of L-strand replication partially overlaps the adjacent tRNA(Cys). The phylogenetic analyses of genes coding for tRNAs and proteins confirm the intermediate phylogenetic position of lungfish between ray-finned fishes and tetrapods. The complete nucleotide sequence of the African lungfish mitochondrial genome was used to estimate which mitochondrial genes are most appropriate to elucidate deep branch phylogenies. Only a combined set of either protein or tRNA mitochondrial genes (but not each gene alone) is able to confidently recover the expected phylogeny among vertebrates that have diverged up to but not over approximately 400 mya.

Amino Acid Sequence↗

The complete mitochondrial DNA sequence of the bichir (Polypterus ornatipinnis), a basal ray-finned fish: ancient establishment of the consensus vertebrate gene order.

The evolutionary position of bichirs is disputed, and they have been variously aligned with ray-finned fish (Actinopterygii) or lobe-finned fish (Sarcopterygii), which also include tetrapods. Alternatively, they have been placed into their own group, the Brachiopterygii. The phylogenetic position of bichirs as possibly the most primitive living bony fish (Osteichthyes) made knowledge about their mitochondrial genome of considerable evolutionary interest. We determined the complete nucleotide sequence (16,624 bp) of the mitochondrial genome of a bichir, Polypterus ornalipinnis. Its genome contains 13 proteincoding genes, 22 tRNAs, two rRNAs and one major noncoding region. The genome's structure and organization show that this is the most basal vertebrate that conforms to the consensus vertebrate mtDNA gene order. Bichir mitochondrial protein-coding and ribosomal RNA genes have greater sequence similarity to ray-finned fish than to either lamprey or lungfish. Phylogenetic analyses suggest the bichir's placement as the most basal living member of the ray-finned fish and rule out its classification as a lobefinned fish. Hence, its lobe-fins are probably not a shared-derived trait with those of lobe-finned fish (Sarcopterygii).

Amino Acid Sequence↗

Phylogenetic performance of mitochondrial protein-coding genes in resolving relationships among vertebrates.

A large number of studies in evolutionary biology utilize phylogenetic information obtained from mitochondrial DNA. Researchers place trust in this molecule and expect it generally to be a reliable marker for addressing questions ranging from population genetics to phylogenies among distantly related lineages. Yet, regardless of the phylogenetic method and weighting treatment, individual mitochondrial genes might potentially produce misleading evolutionary inferences and hence might not constitute an adequate representation neither of the entire mitochondrial genome nor of the evolutionary history of the organisms from which they are derived. We investigated the performance of all mitochondrial protein-coding genes to recover two expected phylogenies of tetrapods and mammals. According to these tests, mitochondrial protein-coding genes can be roughly classified into three groups of good (ND4, ND5, ND2, cytb, and COI), medium (COII, COIII, ND1, and ND6), and poor (ATPase 6, ND3, ATPase 8, and ND4L) phylogenetic performers in recovering these expected trees among phylogenetically distant relatives. How general our findings are is unclear. Simple length differences and rate differences between these genes cannot account for their different phylogenetic performance. The phylogenetic performance of these mitochondrial genes might depend on various factors that play a role in determining the probability of discovering the correct phylogeny such as the density of lineage creation events in time, the phylogenetic "depth" of the question, lineage-specific rate heterogeneity, and the completeness of taxa representation.

Adenosine Triphosphatases↗

The complete nucleotide sequence of the mitochondrial DNA genome of the rainbow trout, Oncorhynchus mykiss.

The complete nucleotide sequence of the mitochondrial DNA of the rainbow trout, Onchorynchus mykiss, has been determined. The total length of the molecule is 16,660 bp. The rainbow trout mitochondrial DNA has the same organization described in eutherian mammals, the clawed frog (Xenopus laevis), and the two fish species, Oriental stream loach (Crossotoma lacustre) and carp (Cyprinus carpio). Alignment and comparison of the deduced amino acid sequences of the 13 proteins encoded by rainbow trout and other vertebrate mitochondrial genomes allowed us to estimate that COI is the most conserved mitochondrial subunit (amino acid identity ranging from 85.6% to 94.8%) whereas ATPase 8 is the most variable one (amino acid identity ranging from 30.8% to 70.4%). Putative secondary structures for the 22 tRNAs found in the molecule are given along with an extensive comparison of tRNA sequences among representative species of each major group of vertebrates. In this sense, an unusual cloverleaf structure for the tRNASer(AGY) is proposed. A stem-loop structure inferred for the origin of the L-strand replication (OL) and the presence of a large polycytidine tract in the OL loop is described. The existence of this stretch instead of the usual T-rich sequence reported so far in mammal mtDNAs is explained in terms of a less-strict template dependence of the RNA primase involved in the initiation of L-strand replication.

Animals↗

Revised dinoflagellate phylogeny inferred from molecular analysis of large-subunit ribosomal RNA gene sequences.

The nucleotide sequence analysis of the PCR products corresponding to the variable large-subunit rRNA domains D1, D2, D9, and D10 from ten representative dinoflagellate species is reported. Species were selected among the main laboratory-grown dinoflagellate groups: Prorocentrales, Gymnodiniales, and Peridiniales which comprise a variety of morphological and ecological characteristics. The sequence alignments comprising up to 1,000 nucleotides from all ten species were employed to analyze the phylogenetic relationships among these dinoflagellates. Maximum parsimony and neighbor-joining trees were inferred from the data generated and subsequently tested by bootstrapping. Both the D1/D2 and the D9/D10 regions led to coherent trees in which the main class of dinoflagellates. Dinophyceae, is divided in three groups: prorocentroid, gymnodinioid, and peridinioid. An interesting outcome from the molecular phylogeny obtained was the uncertain emergence of Prorocentrum lima. The molecular results reported agreed with morphological classifications within Peridiniales but not with those of Prorocentrales and Gymnodiniales. Additionally, the sequence comparison analysis provided strong evidence to suggest that Alexandrium minutum and Alexandrium lusitanicum were synonymous species given the identical sequence they shared. Moreover, clone Gg1V, which was determined Gymnodinium catenatum based on morphological criteria, would correspond to a new species of the genus Gymnodinium as its sequence clearly differed from that obtained in G. catenatum. The sequence of the amplified fragments was demonstrated to be a valuable tool for phylogenetic and taxonomical analysis among these highly diversified species.

Animals↗

Analysis of the transcription products of the rainbow trout (Oncorynchus mykiss) liver mitochondrial genome: detection of novel mitochondrial transcripts.

We have isolated and, by electrophoresis, using agarose slab gels in the presence of methylmercury hydroxide, analyzed the mitochondrial RNA content of the liver of rainbow trout. The RNAs corresponding to most of the mitochondrial DNA-encoded genes have been identified. Furthermore, among the transcription products we have also identified the nature of the RNA 8 previously described in human mitochondria, and detected a novel transcript that may represent the mRNA for ND6.

Animals↗

Nucleotide sequence of the sheep mitochondrial DNA D-loop and its flanking tRNA genes.

The nucleotide sequence of the sheep mitochondrial DNA displacement-loop (D-loop) region and its flanking tRNA genes has been determined. Several conserved motifs among mammals have been identified along the 1189-bp sequence of the sheep control region: ten termination-associated sequences (TASs) and one conserved sequence block (CSB-1). CSB-2 and CSB-3, which are frequently found in most species, are not present in the sheep D-loop, which shows instead a short direct repeat at their usual localization. A long polypyrimidine tract between CSB-1 and the tRNA(Phe) gene is also present. Furthermore, the sheep mtDNA D-loop region displays tandem repeats in the left domain (adjacent to the tRNA(Pro) gene) comprising three different termination-associated sequences (TAS-5, TAS-6 and TAS-7).

Animals↗

Rapid and sensitive detection of the bovine viral diarrhea virus genome in semen.

A reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed for the detection of bovine viral diarrhea virus (BVDV) in cell culture supernatant and in bovine semen. Several sets of primers, PCR conditions and extraction methods were examined to optimize the procedure. A set of primers designed to amplify a highly conserved portion of the p80 gene from BVDV (corresponding to NADL strain sequence from bp 6668 to 7107), was demonstrated to be the most effective. These oligonucleotide primers consistently amplify a 440-bp fragment from several non-cytopathic and cytopathic biotypes of BVDV. The viral origin of the PCR products was assessed by sequencing. The introduction of a Sephacryl S-400 chromatography step to remove seminal inhibitors prior to RNA extraction permitted RT-PCR detection of BVDV in raw and extended semen samples. A maximum sensitivity of 0.4 TCID50 was achieved with this method using RNA extracted from tissue supernatants. This RT-PCR assay may be a useful tool for the detection of BVDV in semen of persistently infected bulls.

Animals↗

Direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus by reverse polymerase chain reaction (RT-PCR).

A method for direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus was developed, based on reverse transcription of the viral RNA coupled to DNA amplification by polymerase chain reaction. A set of primers was designed from Lelystad virus sequence within ORF 7 encoding nucleocapsid protein. From seven Spanish field isolated strains the 312 bp amplified fragment was cloned and sequenced. Alignment with Lelystad virus sequence revealed a 96-97% homology. A maximum sensitivity of 6.7 TCID50 was achieved with the reported procedure in experimentally infected swine alveolar macrophages cultures. The sensitivity obtained in crude clinical samples from experimentally infected 3-weeks old pigs was approximately 10(2) TCID50. High specificity for the PRRS virus was demonstrated for the method, as none of the seven common swine virus assayed rendered DNA amplification product.

Animals↗

Lipogenic activities in rat liver are subjected to circadian rhythms.

Diurnal variations were investigated in the activities of the key lipogenic enzymes in rat liver under standard experimental conditions. Fatty acid synthetase, acetyl-CoA-carboxylase, and ATP-citrate lyase showed sinusoidal circadian rhythms (statistically sustained), reaching maximum activity at night and minimum during the light period, with a fold increase value of 1.7, 2.2 and 3.2, respectively. Although a non-sinusoidal circadian rhythm was observed in the malic enzyme activity, statistically different time-dependent activity values were detected throughout a day. The observed rhythms run in parallel with that previously reported for glucose-6-phosphate dehydrogenase, suggesting a whole circadian regulation for rat liver lipogenesis.

ATP Citrate (pro-S)-Lyase↗

High resolution of proteins by double-inverted gradient polyacrylamide gel electrophoresis (DG-PAGE).

We have designed a new method for high resolution electrophoretic separation of proteins that have similar molecular weights. The proteins migrate first through a conventional gradient gel, in which molecular friction increases as pore size decreases. The proteins then enter an inverted sodium dodecyl sulfate (SDS) gradient gel in which friction decreases; thus, smaller molecules gradually migrate faster and achieve improved separation from larger molecules, which remain near the border between the two gels. We therefore call this technique double-inverted gradient polyacrylamide gel electrophoresis (DG-PAGE). This technique was used to resolve mixtures of aldolase, horseradish peroxidase precursors, glucose 6-phosphate dehydrogenase and pyruvate kinase. By comparison with other established methods, we show that DG-PAGE has a higher resolving power, which achieves clear separation of proteins differing as little as 0.5 kDa in molecular weight.

Animals↗