Molecular cloning of thehom—thrC—thrB cluster fromBacillus sp. ULM1: Expression of thethrC gene inEscherichia coli and corynebacteria,and evolutionary relationships of the threonine genes

A 6.5 kb DNA fragment containing the gene (thrC) encoding threonine synthase, the last enzyme of the threonine biosynthetic pathway, has been cloned from the DNA ofBacillus sp. ULM1 by complementation ofEscherichia coli andBrevibacterium lactofermentum thrC auxotrophs. Complementation studies showed that thethrB gene (encoding homoserine kinase) is found downstream from thethrC gene, and analysis of nucleotide sequences indicated that thehom gene (encoding homoserine dehydrogenase) is located upstream of thethrC gene. The organization of this cluster of genes is similar to theBacillus subtilis threonine operon (hom—thrC—thrB). An 1.9 kbBclI, fragment from theBacillus sp. ULM1 DNA insert that complementedthrC mutations both inE. coli and in corynebacteria was sequenced, and an ORF encoding a protein of 351 amino acids was found corresponding to a protein of 37462 Da. ThethrC gene showed a low G+C content (39.4%) and the encoded threonine synthase is very similar to theB. subtilis enzyme. Expression of the 1.9 kbBclI DNA fragment inE. coli minicells resulted in the formation of a 37 kDa protein. The upstream region of this gene shows promoter activity inE. coli but not in corynebacteria. A peptide sequence, including a lysine that is known to bind the pyridoxal phosphate cofactor, is conserved in all threonine synthase sequences and also in the threonine and serine dehydratase genes. Amino acid comparison of nine threonine synthases revealed evolutionary relationships between different groups of bacteria. Dedicated to Dr. J. Spížek on the occasion of his 60th birthday     

Mothers that produce sons and daughters are genetically different in red deer

Sex ratio theory, and in particular Fisher principle, assumes parental control over the sex of offspring through the action of autosomal genes with Mendelian segregation. In spite of the importance of Fisher's principle in evolutionary biology, the number of studies looking for possible loci involved in sex ratio bias is, at best, very low. Newly developed genetic tools frequently allow evolutionary biologists to manage genetic data. Here we encourage the application of association tools to databases that include genetic information for autosomal loci and offspring sex to improve our knowledge on sex ratio evolution. As an example we use microsatellite markers to scan autosomal chromosomes and look for linked genetic regions associated with offspring sex in red deer (Cervus elaphus). We found a microsatellite marker (CelJP38) mapped in chromosome 27 for which females producing sons and daughters were genetically different. To the best of our knowledge, this is the first study that shows a genetic signal that points out an association between mother genotype and offspring sex in natural populations of a mammal.     

Factors affecting the production of SCEs by maleic hydrazide in root-tip chromosomes of Allium cepa

We have investigated the influence of pH on the induction of chromatid-type aberrations and sister-chromatid exchanges (SCEs) by maleic hydrazide (MH) in root-tip cells of Allium cepa. For both cytogenetic endpoints, the lower the pH of the treatment solution, the higher were the frequencies of chromosome alterations detected at metaphase. We have further studied the persistence of lesions giving rise to SCEs during successive cell cycles, as well as the influence of BrdU concentration in the post-treatment medium on the yield of MH-induced SCEs. Our results suggest that the cytogenetic action of MH in many respects resembles that of bifunctional alkylating agents.     

Influence of Residue 22 on the Folding, Aggregation Profile, and Toxicity of the Alzheimer's Amyloid β Peptide

Several biophysical techniques have been used to determine differences in the aggregation profile (i.e., the secondary structure, aggregation propensity, dynamics, and morphology of amyloid structures) and the effects on cell viability of three variants of the amyloid β peptide involved in Alzheimer's disease. We focused our study on the Glu²² residue, comparing the effects of freshly prepared samples and samples aged for at least 20 days. In the aged samples, a high propensity for aggregation and β-sheet secondary structure appears when residue 22 is capable of establishing polar (Glu²² in wild-type) or hydrophobic (Val²² in E22V) interactions. The Arctic variant (E22G) presents a mixture of mostly disordered and α-helix structures (with low β-sheet contribution). Analysis of transmission electron micrographs and atomic force microscopy images of the peptide variants after aging showed significant quantitative and qualitative differences in the morphology of the formed aggregates. The effect on human neuroblastoma cells of these Aβ_12-28 variants does not correlate with the amount of β-sheet of the aggregates. In samples allowed to age, the native sequence was found to have an insignificant effect on cell viability, whereas the Arctic variant (E22G), the E22V variant, and the slightly-aggregating control (F19G-F20G) had more prominent effects.     

Dynamic analysis of cockroach giant interneuron activity evoked by forced displacement of cercal thread-hair sensilla

This investigation involved extracellular recordings of cockroach abdominal giant interneuron (GI) action potentials evoked by cercal “threadlike” hair sensilla (THS) stimulation with a galvanometric device, by controlled displacements of about seven THS. Small and large GIs, distinguished by their amplitudes, were studied simultaneously. Only the small GIs were spontaneously active. Responses to sine, pulse, and ramp stimulation of sensilla produced phasic responses in both GI types. Some GIs were directionally sensitive and had shorter response latencies in the direction of best sensitivity while others were omnidirectional. Contralateral stimulation decreased responses to homolateral stimuli. In experiments using paired pulses (less than 50-ms intervals) there is a period of hyperexcitability, in large GIs, in which the response to the second stimulus is greater. Repeated stimulation caused an exponential decline in the response which was steeper in all GIs at higher stimulating frequencies and had a faster time course in large GIs. Because of this last property GIs function as low-pass filters limiting the flow of information, with large GIs having a lower frequency “cutoff” than smaller GIs.     

Mitomycin C, 4-nitroquinoline-1-oxide and ethyl methanesulfonate induced long-lived lesions in DNA which result in SCEs during successive cell cycles in human lymphocytes

The present study was carried out in order to analyze how persistent the lesions in DNA are which elicit sister-chromatid exchanges (SCEs), induced by three different chemical agents, mitomycin C (MMC), 4-nitroquinoline-1-oxide (4NQO) and ethyl methanesulfonate (EMS), in proliferating human lymphocytes. Cells were exposed to the mutagens for 1 h just before starting bromodeoxyuridine substitution and SCEs were examined in third-cycle metaphases showing three-way-differential staining, by means of our previously standardized method. The results show that, in spite of the fact that these three compounds have different modes of action, the lesions induced by all of them seem to be capable of persisting in DNA and eliciting SCEs for at least three successive cell cycles.