Ana Camacho

Suppressor-sensitive mutants and genetic map of Bacillus subtilis bacteriophage Φ29

Abstract Suppressor-sensitive mutants of phage Φ29 have been isolated and classified in 13 complementation groups. These mutants have been complemented with the temperature-sensitive mutants of Talavera et al. (1971) and mutant tsQ1360 reported in this paper; all together they make a total of 17 complementation groups. Two- and three-factor crosses have been carried out with sus and ts mutants, and 16 cistrons have been arranged in a linear genetic map. This map, integrating suppressor- and temperature-sensitive mutants, has a total of 24.2 recombination units.

Conservation to the Rescue of Taxonomy

Biodiversity studies and conservation measures depend on good and up-to-date taxonomic data. General faunistic lists are the result of long periods of sampling. How many species on a global list are actually living in an area can only be answered by updating inventories. The progressive disappearance of taxonomic specialists and the undervaluation of their work is not only unjustified, but could lead to specialists from other disciplines working with meaningless data.

A precursor of the neck appendage protein of B. subtilis phage Φ 29

Comision Asesora para la Investigacion Cientifica y Fondo Nacional para la Formacion de Personal Investigador

Characterization of deoxyuridine 5'-triphosphate nucleotidohydrolase from Trypanosoma cruzi.

We report the cloning and kinetic characterization of Trypanosoma cruzi deoxyuridine 5′‐triphosphate nucleotidohydrolase (dUTPase) whose coding sequence was isolated by genetic complementation in Escherichia coli. The deduced amino acid sequence was similar to Leishmania major dUTPase although it exhibits an amino acid insertion which is sensitive to protease inactivation. The catalytically active species of the enzyme is a dimer and a detailed kinetic characterization showed that it is highly specific for dUTP and dUDP. The general observation that dUTPases from the Trypanosomatidae differ in sequence, conformation and substrate specificity suggests that a different family of dUTPases exists in certain organisms, which may be exploited as drug targets against infectious diseases.

Mechanism for the switch of φ29 DNA early to late transcription by regulatory protein p4 and histone-like protein p6

Bacteriophage φ29 gene expression takes place from four major promoters, three of them (A2b, A2c and A3) clustered within 219 bp at a central region of the genome. Transcription regulation of these promoters involves both a highly specific DNA‐binding protein (p4) and a low specificity DNA‐binding protein (p6) functionally related to prokaryotic histone‐like proteins. Protein p6 forms extended oligomeric arrays along the phage DNA. In contrast, protein p4 binds specifically upstream of late promoter A3 and early promoter A2c. We have analysed the concomitant binding of p6 and p4 and found that the proteins cooperate with each other in the binding to the central region of the genome, resulting in a ternary p4–p6–DNA complex that affects local DNA topology. Through this complex, protein p6 exerts a direct role in the repression of promoter A2c, impeding unwinding of the DNA strands needed for open complex formation. In contrast, protein p6 functions by reinforcing the positioning of protein p4 in the repression of promoter A2b and activation of promoter A3, thereby facilitating p4‐mediated transcription regulation.

A new genus of Parabathynellidae (Crustacea: Bathynellacea) in New South Wales, Australia

A new genus and species of the family Parabathynellidae, Octobathynella peelensis gen. nov. sp. nov., is described from New South Wales, Australia. The new genus displays several exclusive characters: a very large and distinctive male thoracopod VIII with one crest-like projection containing two lobules on the basipod; and four aesthetascs on segments six and seven of the antennule. It also has several combinations of characters that make it unique in the Parabathynellidae, specifically that: the antennule is eight-segmented (a new character in the Australian species); there are seven segments in the antenna; the labrum has 18–20 teeth; the exopod of thoracopod I has three to four segments and the exopod of thoracopod II has four segments; the endopod has one dorsal seta on the first and second segments and the epipod is absent on thoracopod I; pleopods are absent; the endopod of the male thoracopod VIII has two setae and the exopod has setules; there are 10–12 spines on the sympod, three or four spines on the endopod and five setae on the exopod of the uropod. The new genus and species is placed into context with all known Bathynellacea in Australia, and the biogeographic patterns are discussed for this ancient group of subterranean crustaceans.

Phylogenetical review and biogeographic remarks on the interstitial and subterranean freshwater iberobathynells (Crustacea, Syncarida, Parabathynellidae)

In order to infer evolutionary patterns within iberobathynells (Parabathynellidae, Bathynellacea) a complete morphological description and population variability study of the recently described genera Guadalopebathynella Camacho and Serban, 1998 and Hexaiberobathynella Camacho and Serban, 1998, endemic to the Iberian Peninsula, are presented. We compared 14 different populations of the Hexaiberobathynella mateusi (Galhano, 1967), 12 in Spain and two in Portugal, with a new population recently ascribed to the poorly known Hexaiberobathynella hortezuelensis Camacho and Serban, 1998. All these genera show autapomorphic characteristics and a unique combination of shared derived characters that are the basis of their monophyletic status as a tribe. The origin and distribution of the genera of freshwater interstitial (stygobiont) syncarids of the tribe Iberobathynellini (Crustacea), endemic to North America (California and Texas), Iberian Peninsula, Balearic Islands and North Africa (Morocco) are considered within the scope of evolutionary biogeography. Both plate tectonics and the Boutin-Coineau two-step model of colonization and evolution, the second phase of which represents vicariance caused by the Tethys regressions, are used to understand the evolutionary history of freshwater stygobiont iberobathynell species. Correlation between time of the last marine regression for each generic range today, and order of derivation in the phylogenetic trees show it is not significant. From these results we cannot reject the possibility that the evolutionary clade divergence within iberobathynells is independent of sea level changes and therefore we can neither accept nor reject their correlation.

Antigen selection and presentation to protect against transmissible gastroenteritis coronavirus

Abstract The antigenic structure of the S glycoprotein of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) has been determined and correlated with the physical structure. Four antigenic sites have been defined (A, B, C, and D). The sites involved in the neutralization of TGEV are: A, D, and B, sites A and D being antigenically dominant for TGEV neutralization in vitro. These two sites have specific properties of interest: site A is highly conserved and is present in coronaviruses of three animal species, and site D can be represented by synthetic peptides. Both sites might be relevant in protection in vivo. PRCV does not have sites B and C, due to a genomic deletion. Complex antigenic sites, i.e., conformation and glycosylation dependent sites, have been represented by simple mimotopes selected from a library expressing recombinant peptides with random sequences, or by anti-idiotypic internal image monoclonal antibodies. An epidemiological tree relating the TGEVs and PRCVs has been proposed. The estimated mutation fixation rate of 7 ± 2 × 10−4 substitutions per nucleotide and year indicates that TGEV related coronaviruses show similar variability to other RNA viruses. In order to induce secretory immunity, different segments of the S gene have been expressed using avirulent forms of Salmonella typhimurium and adenovirus. These vectors, with a tropism for Peyers patches may be ideal candidates in protection against TGEV.

A precise DNA bend angle is essential for the function of the phage φ29 transcriptional regulator

Bacteriophage φ29 protein p4 is essential for the regulation of the switch from early to late phage transcription. The protein binds to two regions of the phage genome located between the regulated promoters. Each region contains two inverted repeats separated by 1 bp. We used circular permutation assays to study the topology of the DNA upon binding of the protein and found that p4 induced the same extent of bending independent of the topology of the binding region. In addition, the results revealed that the p4-induced bending is not dependent on the affinity to the binding site but is intrinsic to p4 binding. Independent binding sites were identified through the characterization of the minimal sequence required for p4 binding. The protein has different affinity for each of its binding sites, with those overlapping the A2c and A2b promoter cores (sites 1 and 3), having the highest affinity. The functionality of the p4 binding sites and the contribution of p4-mediated promoter restructuring in transcription regulation is discussed.

Four new Species of Groundwater Crustaceans (Syncarida, Bathynellacea, Parabathynellidae) Endemic to the Iberian Peninsula

Four new species of the genus Iberobathynella from the Iberian Peninsula are described and complementary diasgnostic features are provided to the original description of Iberobathynella (Asturibathynella) cavadoensis. Two of the new species were found in the interstitial environment (gravel bank) of two streams in Portugal (in Ponte de Lima and Coimbra) and two others in Spain. One of the latter was in a subterranean river in the largest cave in Spain (Ojo Guareña Cave, Burgos, 100 km in length), and the other was in the gravel bank of a small stream in Lora del Río (Sevilla). Specimens of I. (A.) cavadoensis from the type locality (Cavado river in Barcelos, Portugal) are examined in this paper. Of the 22 species of iberobathynells so far described none show the combinations of characters present in the four new species described here. These new character combinations will be very useful in resolving current phylogenetic problems in this group.