Maintenance of high levels of allelic variation in spite of a severe bottleneck in population size: the brown bear (Ursus arctos) in the Western Carpathians

Genetic variation in an isolated brown bear population of the Western Carpathians was studied by electrophoretic analysis of 51 presumptive allozyme loci. In spite of a severe population bottleneck at the beginning of the 1930s (40 survivors), average heterozygosity (H=5.3%) was within the range commonly found in mammals and the mean number of alleles per polymorphic locus (A_p=3, over five polymorphic loci) was very high. Possible reasons for the maintenance of high allelic variation are discussed.     

Scarless skin wound repair in the fetus.

The ability of a fetus to heal without scar formation depends on its gestational age at the time of injury and the size of the wound defect. In general, linear incisions heal without scar until late in gestation whereas excisional wounds heal with scar at an earlier gestational age. The profiles of fetal proteoglycans, collagens, and growth factors are different from those in adult wounds. The less-differentiated state of fetal skin is probably an important characteristic responsible for scarless repair. There is minimal inflammation in fetal wounds. Fetal wounds are characterized by high levels of hyaluronic acid and its stimulator(s) with more rapid, highly organized collagen deposition. The roles of peptide growth factors such as transforming growth factor-beta and basic fibroblast growth factor are less prominent in fetal than in adult wound healing. Platelet-derived growth factor has been detected in scarless fetal skin wounds, but its role is unknown. An understanding of scarless tissue repair has possible clinical application in the modulation of adult fibrotic diseases and abnormal scar-forming conditions.     

Use of polymerase chain reaction and electroporation of Escherichia coli to monitor the persistence of extracellular plasmid DNA introduced into natural soils.

A modified protocol for DNA amplification by polymerase chain reaction (PCR) coupled with laser densitometric determination of the amount of PCR products, which allowed quantitation of target sequence numbers in soil extracts, was developed. The method was applied to monitor target loss during incubation of purified plasmid DNA in natural nonsterile soils. It revealed soil-specific kinetics of target loss. After 60 days, 0.2, 0.05, and 0.01% of the initially added nahA genes on plasmids were detectable by PCR in a loamy sand soil, a clay soil, and a silty clay soil, respectively. Electroporation of Escherichia coli was used in parallel to quantitate plasmid molecules in soil extracts by their transforming activity. It was found that transformation by electroporation was about 20 times more efficient and much less inhibited by constituents of soil extracts than transformation of Ca(2+)-treated cells (G. Romanowski, M.G. Lorenz, G. Sayler, and W. Wackernagel, Appl. Environ. Microbiol. 58:3012-3019, 1992). By electroporation, greater than 10,000-fold plasmid loss was monitored in nonsterile soils. Transforming activity was found up to 60 days after inoculation of the soils. The studies indicate that PCR and electroporation are sensitive methods for monitoring the persistence of extracellular plasmid DNA in soil. It is proposed that plasmid transformation by electroporation can be used for the monitoring in soil and other environments of genetically engineered organisms with recombinant plasmids. The data suggest that genetic material may persist in soil for weeks and even for months after its release from cells.     

Complete ammonia oxidization in agricultural soils: High ammonia fertilizer loss but low N2O production

The contribution of agriculture to the sustainable development goals requires climate-smart and profitable farm innovations. Increasing the ammonia fertilizer applications to meet the global food demands results in high agricultural costs, environmental quality deterioration, and global warming, without a significant increase in crop yield. Here, we reported that a third microbial ammonia oxidation process, complete ammonia oxidation (comammox), is contributing to a significant ammonia fertilizer loss (41.9 ± 4.8%) at the rate of 3.53 ± 0.55 mg N kg⁻¹ day⁻¹ in agricultural soils around the world. The contribution of comammox to ammonia fertilizer loss, occurring mainly in surface agricultural soil profiles (0–0.2 m), was equivalent to that of bacterial ammonia oxidation (48.6 ± 4.5%); both processes were significantly more important than archaeal ammonia oxidation (9.5 ± 3.6%). In contrast, comammox produced less N₂O (0.98 ± 0.44 μg N kg⁻¹ day⁻¹, 11.7 ± 3.1%), comparable to that produced by archaeal ammonia oxidation (16.4 ± 4.4%) but significantly lower than that of bacterial ammonia oxidation (72.0 ± 5.1%). The efficiency of ammonia conversion to N₂O by comammox (0.02 ± 0.01%) was evidently lower than that of bacterial (0.24 ± 0.06%) and archaeal (0.16 ± 0.04%) ammonia oxidation. The comammox rate increased with increasing soil pH values, which is the only physicochemical characteristic that significantly influenced both comammox bacterial abundance and rates. Ammonia fertilizer loss, dominated by comammox and bacterial ammonia oxidation, was more intense in soils with pH >6.5 than in soils with pH <6.5. Our results revealed that comammox plays a vital role in ammonia fertilizer loss and sustainable development in agroecosystems that have been previously overlooked for a long term.     

Protein folding in the cell: molecular chaperones pave the way

In vitro, many unfolded polypeptides are able to fold to the native state spontaneously, indicating that the amino acid sequence of a protein contains all the information necessary to specify its three-dimensional conformation. It had been assumed that protein folding in vivo also generally occurs in a spontaneous process. This view has changed only recently due to the discovery of a number of proteins, now commonly called 'molecular chaperones', which are essential for cellular protein folding and occur ubiquitously in eubacteria, archaebacteria and in eukaryotic cells.     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling.

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.     

Zolpidem Displays Heterogeneity in Its Binding to the Nonhuman Primate Benzodiazepine Receptor In Vivo

Abstract: The distinctive pharmacological activity of zolpidem in rats compared with classical benzodiazepines has been related to its differential affinity for benzodiazepine receptor (BZR) subtypes. By contrast, in nonhuman primates the pharmacological activity of zolpidem was found to be quite similar to that of classical BZR agonists. In an attempt to explain this discrepancy, we examined the ability of zolpidem to differentiate BZR subtypes in vivo in primate brain using positron emission tomography. The BZRs were specifically labeled with [¹¹C]flumazenil. Radiotracer displacement by zolpidem was monophasic in cerebellum and neocortex, with in vivo Hill coefficients close to 1. Conversely, displacement of [¹¹C]flumazenil was biphasic in hippocampus, amygdala, septum, insula, striatum, and pons, with Hill coefficients significantly smaller than 1, suggesting two different binding sites for zolpidem. In these cerebral regions, the half-maximal inhibitory doses for the high-affinity binding site were similar to those found in cerebellum and neocortex and ～100-fold higher for the low-affinity binding site. The low-affinity binding site accounted for <32% of the specific [¹¹C]-flumazenil binding. Such zolpidem binding characteristics contrast with those reported for rodents, where three different binding sites were found. Species differences in binding characteristics may explain why zolpidem has a distinctive pharmacological activity in rodents, whereas its pharmacological activity in primates is quite similar to that of classical BZR agonists, except for the absence of severe effects on memory functions, which may be due to the lack of substantial zolpidem affinity for a distinct BZR subtype in cerebral structures belonging to the limbic system.     

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.