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. Correspondence to: J. Dopazo     

A Drosophila hsp70 gene contains long,antiparallel, coupled open reading frames (LAC ORFs) conserved in homologous loci

A clone isolated from a Drosophila auraria heat-shock cDNA library presents two long, antiparallel, coupled (LAC) open reading frames (ORFs). One strand ORF is 1,929 nucleotides long and exhibits great identity (87.5% at the nucleotide level and 94% at the amino acid level) with the hsp70 gene copies of D. melanogaster, while the second strand ORF, in antiparallel in-frame register arrangement, is 1,839 nucleotides long and exhibits 32% identity with a putative, recently identified, NAD⁺-dependent glutamate dehydrogenase (NAD⁺-GDH). The overlap of the two ORFs is 1,824 nucleotides long. Computational analysis shows that this LAC ORF arrangement is conserved in other hsp70 loci in a wide range of organisms, raising questions about possible evolutionary benefits of such a peculiar genomic organization.Correspondence to: Z.G. Scouras     

Mutant Escherichia coli penicillin acylase with enhanced stability at alkaline pH

Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6-aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide-directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431-Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half-life of the mutant enzyme when stored at pH 8.5 as compared with the wild-type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH-stability profile. (c) 1995 John Wiley & Sons, Inc.     

Copy number determination of different derivatives of the streptomycete mini-plasmid pSLG33

Copy numbers of the streptomycete plasmid vector pRS410 and five other recombinant plasmid derivatives of the original cryptic streptomycete plasmid pSLG33 were determined using calibrated laser densitometry. DNA preparations, electrophoretically separated on agarose gels, were stained with ethidium bromide, photographed and the negatives were subsequently scanned in a laser densitometer. The pSLG33 replicon is very stable, as no effect of the selective pressure was observed. It is a multicopy plasmid with up to 220 detected copies per chromosome. The use of deletion and/or insertion mutants allowed us to define two regions of the pSLG33 molecule involved in the control of plasmid replication.     

Staphylococcus aureus in chronic and recurrent infections

Ninety-fourStaphylococcus aureus strains isolated from chronic and recurrent skin and respiratory tract infections were investigated for several virulence factor expressions. Production of protein A was noticed in all of the tested strains in amounts from less than 0.1 to more than 2.5 ng per 10⁶ bacterial cells. The percentage of the extracellularly produced protein A was found to lie between 4.5 and 27.8%. Two strains (both from the respiratory tract) produced more than 50 % of protein A in the extracellular form and one strain did not produce any detectable amount of the extracellular protein A; 99 % of the tested strains produced the clumping factor, 96% staphylocoagulase, 79 % staphylokinase and 90 % gelatinolytic activity; 79 % produced α-toxin exclusively or in combination with δ- or β-toxin; 8 % of strains produced β-toxin. There were differences in β-toxin production between strains from the respiratory tract (5 %) and skin infections (25 %). δ-Toxin was produced by 53 % of the strains. In each of the tested strains a complex of virulence factors was detected. The importance of inactivated extracellular products (especially α- and δ-toxin and in the case of skin infections also β-toxin) as components of staphylococcal whole-cell vaccine was suggested. Dedicated to Professor C. John on the occasion of his 75th birthday     

Biodiversity of sweetpotato (iPomoea batatas,Convolvulaceae) in Southern Mexico

Between 1987 and 1992 the phytogeographic region of southern Mexico was explored during three collecting trips made in search of cultivated and wild germplasm of the sweetpotato (Ipomoea batatas). The first trip was made in 1987, when we collected wild species in Ipomoea section Batatas found in the southeastern and southwestern regions of Mexico. A second trip was made in 1990, when we collected accessions of the cultivated species as well as wild species in the southeast, southwest and northeast. The third and final trip was oriented at identification, characterization and collecting seeds in the ecological niches ofI.tabascana andI.umbraticola. As a result of the three trips we collected 165 accessions of cultivated and wild germplasm with populations dispersed in 147 localities in 15 states of the Mexican region: Veracruz, Tabasco, Campeche, Chiapas, Oaxaca, Yucatán, Guerrero, Michoacán, San Luis Potosí, Hidalgo, Querétaro, Tamaulipas, Guanajuato, Puebla and México. Of the total accessions some 64 (38.3%) were of the cultivated species including nine accessions of feral material, and 103 accessions (61.7%) were of wild species made up of 59 accessions of seven species in the section Batatas, 37 of other species in the family Convolvulaceae, and seven yet to be determined. We have identified the largest genetic biodiversity in six localities of five states: Tabasco, Oaxaca, Michoacán, San Luis Potosí, and Puebla. Biodiversity maintenance in this region is associated with the day-of-the-dead festivities.     

Further insights on chromosomal pairing of autopolyploids: a triploid and tetraploids of rye

Chromosomal pairing of one triploid and three tetraploid plants of rye, Secale cereale, was analyzed by electron microscopy in surface-spread prophase I nuclei and compared with light microscopic observations of metaphase I cells. Prophase I is characterized by: (i) the weak alignment showed by the three or four unsynapsed or partially homologous synapsed axes; (ii) the low number ber of pairing partner switches (PPSs) displayed by both trivalents and quadrivalents; and (iii) the existence of complex multivalents in which up to 13 chromosomes in the triploid and 22 chromosomes in the tetraploids were involved. However, only few heterologous chromosomal associations were maintained at metaphase I. The results obtained are discussed under the assumptions of the random end pairing model with some modifications.     

P1 clones from Drosophila melanogaster as markers to study the chromosomal evolution of Muller's A element in two species of the obscura group of Drosophila

Thirty P1 clones from the X chromosome (Muller's A element) of Drosophila melanogaster were cross-hybridized in situ to Drosophila subobscura and Drosophila pseudoobscura polytene chromosomes. An additional recombinant phage Dsuby was also used as a marker. Twenty-three (77%) of the P1 clones gave positive hybridization on D. pseudoobscura chromosomes bat only 16 (53%) did so with those of D. subobscura. Eight P1 clones gave more than one hybridization signal on D. pseudoobscura and/or D. subobscura chromosomes. All P1 clones and Dsuby hybridized on Muller's A element (X chromosome) of D. subobscura. In contrast, only 18 P1 clones and Dsuby hybridized on Muller's D element (XR chromosomal arm) of D. pseudoobscura; 4 additional P1 clones hybridized on Muller's D element (XR chromosomal arm) of this species and the remaining P1 clone gave on hybridization signal on each arm of the X chromosome. This latter clone may contain one breakpoint of a pericentric inversion that may account for the interchange of genetic material between Muller's A and D elements in D. pseudoobscura. In contrast to the rare interchange of genetic material between chromosomal elements, profound differences in the order and spacing of markers were detected between D. melanogaster, D. pseudoobscura and D. subobscura. In fact, the number of chromosomal segments delimited by identical markers and conserved between pairwise comparisons is small. Therefore, extensive reorganization within Muller's A element has been produced during the divergence of the three species. Rough estimates of the number of cytologically detectable inversions contributing to differentiation of Muller's A element were obtained. The most reliable of these estimates is that obtained from the D. pseudoobscura and D. melanogaster comparison since a greater number of markers have been mapped in both species. Tentatively, one inversion breakpoint about every 200 kb has been produced and fixed during the divergence of D. pseudoobscura and D. melanogaster.     

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

During early embryogenesis of the nematode Parascaris univalens (2n=2) the processes of chromatin diminution and segregation of the germ and somatic cell lineages take place simultaneously. In this study we analyzed the nucleolar cycle in early embryos, both in germinal and somatic blastomeres, by means of silver staining and antibodies against the nucleolar protein fibrillarin. We observed an identical nucleolar cycle in both types of blastomeres, hence, the chromatin diminution process has no effect on the nucleolar cycle of somatic blastomeres. We report the existence of outstanding differences between this cycle and those previously reported during early embryogenesis of other species. There is a true nucleolar cycle in early embryos that shows a peculiar nucleolar disorganization at prophase, and a preferential localization of prenucleolar bodies only on the euchromatic regions during nucleologenesis. Moreover, fibrillarin does not form a perichromosomal sheath in metaphase or anaphase holocentric chromosomes, probably owing to their special centromeric organization. The number and location of nucleolus organizer regions (NORs) in the chromosomal complement have been determined using silver impregnation, chromomycin A₃/distamycin A staining, and fluorescent in situ hybridization using an rDNA probe. There are only two NORs, one per chromosome, and these are lost in blastomeres after chromatin diminution. Moreover, the constant presence of two nucleoli in somatic blastomeres suggests that NORs are not affected during the fragmentation of euchromatic regions when this process occurs.