Mechanochemical study of conformational transitions and melting of Li-, Na-, K-, and CsDNA fibers in ethanol–water solutions

Highly oriented fibers of Li-, Na-, K-, and CsDNA were prepared with a previously developed wet spinning method. The procedure gave a large number of equivalent fiber bundle samples (reference length, L₀, typically = 12–15 cm) for systematic measurements of the fiber length L in ethanol–water solutions, using a simple mechanochemical set up. The decrease in relative length L/L₀ with increasing ethanol concentration at room temperature gave evidence for the B-A transition centered at 76% (v/v) ethanol for NaDNA fibers and at 80 and 84% ethanol for K- and CsDNA fibers. A smaller decrease in L/L₀ of LiDNA fibers was attributed to the B-C transition centered at 80% ethanol. In a second type of experiment with DNA fibers in ethanol–water solutions, the heat-induced helix–coil transition, or melting, revealed itself in a marked contraction of the DNA fibers. The melting temperature T_m, decreased linearly with increasing ethanol concentration for fibers in the B-DNA ethanol concentration region. In the B-A transition region, Na- and KDNA fibers showed a local maximum in T_m. On further increase of the ethanol concentration, the A-DXA region followed with an even steeper linear decrease in T_m. The dependence on the identity of the counterion is discussed with reference to the model for groove binding of cations in B-DNA developed by Skuratovskii and co-workers and to the results from Raman studies of the interhelical bonds in A-DNA performed by Lindsay and co-workers. An attempt to apply the theory of Chogovadze and Frank-Kamenetskii on DNA melting in the B-A transition region to the curves failed. However, for Na- and KDNA the T_m dependence in and around the A-B transition region could be expressed as a weighted mean value of T_m of A- and B-DNA. On further increase of the ethanol concentration, above 84% ethanol for LiDNA and above about 90% ethanol for Na-, K-, and CsDNA, a drastic change occurred. T_m increased and a few percentages higher ethanol concentrations were found to stabilize the DNA fibers so that they did not melt at all, not even at the upper temperature limit of the experiments (～ 80°C). This is interpreted as being due to the strong aggregation induced by these high ethanol concentrations and to the formation of P-DNA. Many features of the results are compatible with the counterion–water affinity model. In another series of measurements, T_m of DNA fibers in 75% ethanol was measured at various salt concentrations. No salt effect was observed (with the exception of LiDNA at low salt concentrations). This result is supported by calculations within the Poisson–Boltzmann cylindrical cell model. © 1994 John Wiley & Sons, Inc.     

Sperm cell number per bundle in Gnorimus nobilis L. (Coleoptera,Scarabaeidae)

An attempt was made to determine the number of spermatozoa per bundle in a scarabaeoid species Gnorimus nobilis both through the analysis of the premeiotic cytology of germ line cells and by counting the spermatids within a spermiocyst at the beginning of the process of spermiogenesis. The obtained results, taken together, indicate that definitive spermatogonia go through a series of 6 synchronous mitotic divisions before entering meiosis as primary spermatocytes, which in turn produce 256 (2⁸) spermatozoa per bundle after completion of meiosis and spermiogenesis. The obtained data are compared with similar ones for other beetle species belonging to the family Scarabaeidae and the suborder Coleoptera-Polyphaga, respectively. Also, some phylogenetic implications of these data are briefly discussed.     

The Phylogenetic Distribution and Evolution of Enzymes Within the Thymidine Kinase 2-like Gene Family in Metazoa

Deoxyribonucleoside kinases (dNKs) carry out the rate-determining step in the nucleoside salvage pathway within all domains of life where the pathway is present, and, hence, are an indication on whether or not a species/genus retains the ability to salvage deoxyribonucleosides. Here, a phylogenetic tree is constructed for the thymidine kinase 2-like dNK gene family in metazoa. Each enzyme class (deoxycytidine, deoxyguanosine, and deoxythymidine kinases, as well as the multisubstrate dNKs) falls into a monophyletic clade. However, in vertebrates, dCK contains an apparent duplication with one paralog lost in mammals, and a number of crustacean genomes (like Caligus rogercresseyi and Lepeophtheirus salmonis) unexpectedly contain not only the multisubstrate dNKs, related to Drosophila multisubstrate dNK, but also a TK2-like kinase. Additionally, crustaceans (Daphnia, Caligus, and Lepeophtheirus) and some insects (Tribolium, Danaus, Pediculus, and Acyrthosiphon) contain several multisubstrate dNK-like enzymes which group paraphyletically within the arthropod clade. This might suggest that the multisubstrate dNKs underwent multiple rounds of duplications with differential retention of duplicate copies between insect families and more complete retention within some crustaceans and insects. Genomes of several basal animalia contain more than one dNK-like sequence, some of which group outside the remaining eukaryotes (both plants and animals) and/or with bacterial dNKs. Within the vertebrates, the mammalian genomes do not contain the second dCK, while birds, fish, and amphibians do retain it. Phasianidae (chicken and turkey) have lost dGK, while it has been retained in other bird lineages, like zebra finch. Reconstruction of the ancestral sequence between the multisubstrate arthropod dNKs and the TK2 clade of vertebrates followed by homology modeling and discrete molecular dynamics calculations on this sequence were performed to examine the evolutionary path which led to the two different enzyme classes. The structural models showed that the carboxyl terminus of the ancestral sequence is more helical than dNK, in common with TK2, although any implications of this for enzyme specificity will require biochemical validation. Finally, rate-shift and conservation-shift analysis between clades with different specificities uncovered candidate residues outside the active site pocket which may have contributed to differentiation in substrate specificity between enzyme clades.     

Complex Nature of the Genome in a Wine Spoilage Yeast,Dekkera bruxellensis

When the genome organizations of 30 native isolates belonging to a wine spoilage yeast, Dekkera (Brettanomyces) bruxellensis, a distant relative of Saccharomyces cerevisiae, were examined, the numbers of chromosomes varied drastically, from 4 to at least 9. When single gene probes were used in Southern analysis, the corresponding genes usually mapped to at least two chromosomal bands, excluding a simple haploid organization of the genome. When different loci were sequenced, in most cases, several different haplotypes were obtained for each single isolate, and they belonged to two subtypes. Phylogenetic reconstruction using haplotypes revealed that the sequences from different isolates belonging to one subtype were more similar to each other than to the sequences belonging to the other subtype within the isolate. Reanalysis of the genome sequence also confirmed that partially sequenced strain Y879 is not a simple haploid and that its genome contains approximately 1% polymorphic sites. The present situation could be explained by (i) a hybridization event where two similar but different genomes have recently fused together or (ii) an event where the diploid progenitor of all analyzed strains lost a regular sexual cycle, and the genome started to accumulate mutations.Recent achievements in genome sequencing have revealed that gene contents vary among distantly related organisms but are relatively constant among closely related species. For example, among hemiascomycete yeasts, which originated more than 250 million years ago and include well-studied yeasts such as Saccharomyces cerevisiae and Candida albicans (3, 4), an average genome contains approximately 5,000 genes. Approximately one-half of the protein-coding gene families are preserved in all of the yeasts sequenced to date. However, there is a large variation in the gene order and configuration of chromosomes among different species.Chromosome configuration is usually well preserved among populations belonging to the same species. Only rarely do geographically separated populations, for example, Mus musculus (8, 32), differ in the number and form of chromosomes. The mutability of the genome enhances the adaptability of the species, but it also decreases the viability of the new variant. In addition, these changes can preclude successful reproduction and can be a decisive factor in the emergence of new species (2; for a review, see references 6 and 7).Among closely related yeasts belonging to the Saccharomyces sensu stricto clade (including S. cerevisiae), which originated approximately 20 million years ago, the gene contents are relatively similar (13). Their genomes are almost colinear and consist of 16 chromosomes. Some inter- and intraspecific variations are observed predominantly at the chromosome ends (18, 19). Sensu stricto species are semifertile, meaning that they can successfully mate and produce F1 offspring but that the hybrids are largely sterile. It appears that this clade has still not completed the speciation process (7). The relatively low chromosome variability among Saccharomyces sensu stricto yeasts is probably promoted by regular sexual cycles. These yeasts are diploid, but heterozygosity is almost absent because of the homothallic life-style, which enables haploid spores from the same yeast cell to mate. Only for “sterile” hybrids, such as the lager brewing yeast Saccharomyces pastorianus (Saccharomyces carlsbergensis), originating upon the mating of two different species, has a pronounced heterozygosity been observed (14). The parental genomes came from S. cerevisiae and a close relative, Saccharomyces bayanus. A study of allotetraploid hybrids between a diploid S. cerevisiae strain and a diploid S. bayanus strain demonstrated that these hybrids behave essentially as diploids regarding meiosis and sporulation and had 77% spore viability (1, 22). The extent of intra- and interspecific genome variability is not well known for other yeasts, especially among distant relatives of S. cerevisiae. The only well-studied exception is a pathogen, Candida albicans, that is believed to be predominantly asexual. This yeast diverged from the S. cerevisiae lineage prior to the origin of the efficient homothallic life-style (reviewed in reference 25). The genome is diploid and shows a low level of heterozygosity (12), and large variations in the configurations of the chromosomes among different isolates have been reported (reviewed in reference 29).Dekkera bruxellensis is often isolated in wineries and is well known as a major microbial cause of wine spoilage. The lineages of D. bruxellensis and S. cerevisiae separated at approximately the same time as the lineages of S. cerevisiae and C. albicans separated, approximately 200 million years ago (40). However, D. bruxellensis and S. cerevisiae share several characteristics, such as the production of ethanol, the ability to propagate in the absence of oxygen (anaerobic growth), and petite positivity (the ability to produce offspring without mitochondrial DNA [mtDNA]), that are rarely found among other yeasts (16, 20). So far, a sexual cycle in D. bruxellensis has not been found.In this paper, we analyzed the genome structures of 30 isolates of D. bruxellensis originating from different geographical localities around the world. We show that these isolates have different numbers and sizes of chromosomes and also that the numbers of copies of several analyzed genes and their sequences vary. In addition, we could detect heterozygosity in the partial genome sequence of strain Y879.     

Determination of reference genes for circadian studies in different tissues and mouse strains

Background  

Circadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Gene expression studies in these pathologies are often studied in different mouse strains by quantitative real time polymerase chain reaction (qPCR). Selection of reference genes is a crucial step of qPCR experiments. Recent studies show that reference gene stability can vary between species and tissues, but none has taken circadian experiments into consideration.     

Structural insights into substrate traffic and inhibition in acetylcholinesterase

Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.     

Mechanism of stereoselective interaction between butyrylcholinesterase and ethopropazine enantiomers

Stereoselectivity of reversible inhibition of butyrylcholinesterase (BChE; EC 3.1.1.8) by optically pure ethopropazine [10-(2-diethylaminopropyl)phenothiazine hydrochloride] enantiomers and racemate was studied with acetylthiocholine (0.002–250 mM) as substrate. Molecular modelling resulted in the reaction between BChE and ethopropazine starting with the binding of ethopropazine to the enzyme peripheral anionic site. In the next step ethopropazine ‘slides down’ the enzyme gorge, resulting in interaction of the three rings of ethopropazine through π–π interactions with W82 in BChE. Inhibition mechanism was interpreted according to three kinetic models: A, B and C. The models differ in the type and number of enzyme–substrate, enzyme–inhibitor and enzyme–substrate–inhibitor complexes, i.e., presence of the Michaelis complex and/or acetylated BChE. Although, all three models reproduced well the BChE activity in absence of ethopropazine, model A was poor in describing inhibition with ethopropazine, while models B and C were better, especially for substrate concentrations above 0.2 mM. However model C was singled out because it approaches fulfilment of the one step-one event criteria, and confirms the inhibition mechanism derived from molecular modelling. Model C resulted in dissociation constants for the complex between BChE and ethopropazine: 61, 140 and 88 nM for R-enantiomer, S-enantiomer and racemate, respectively. The respective dissociation constants for the complexes between acetylated BChE and ethopropazine were 268, 730 and 365 nM. Butyrylcholinesterase had higher affinity for R-ethopropazine.