Haplotype identity between individuals who share a CFTR mutation allele “identical by descent”: demonstration of the usefulness of the haplotype-sharing concept for gene mapping in real populations

Cystic fibrosis (CF) patients with the A455E mutation, in both the French Canadian and the Dutch population, share a common haplotype over distances of up to 25 cM. French Canadian patients with the 621+1G→T mutation share a common haplotype of more than 14 cM. In contrast, haplotypes containing the ΔF508 mutation show haplotype identity over a much shorter genomic distance within and between populations, probably because of the multiple introduction of this most common mutation. Haplotype analysis for specific mutations in CF or in other recessive diseases can be used as a model for studying the occurrence of genetic drift conditional on gene frequencies. Moreover, from our results, it can be inferred that analysis of shared haplotypes is a suitable method for genetic mapping in general. Received: 30 November 1995 / Revised: 11 April 1996     

Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Continuously Percolated Soil Columns

Although the absence of nitrate formation in grassland soils rich in organic matter has often been reported, low numbers of nitrifying bacteria are still found in these soils. To obtain more insight into these observations, we studied the competition for limiting amounts of ammonium between the chemolithotrophic ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis in the presence of Nitrobacter winogradskyi with soil columns containing calcareous sandy soil. The soil columns were percolated continuously at a dilution rate of 0.007 h^-1, based on liquid volumes, with medium containing 5 mM ammonium and different amounts of glucose ranging from 0 to 12 mM.A. globiformis was the most competitive organism for limiting amounts of ammonium. The numbers of N. europaea and N. winogradskyi cells were lower at higher glucose concentrations, and the potential ammonium-oxidizing activities in the uppermost 3 cm of the soil columns were nonexistent when at least 10 mM glucose was present in the reservoir, although 10⁷ nitrifying cells per g of dry soil were still present. This result demonstrated that there was no correlation between the numbers of nitrifying bacteria and their activities. The numbers and activities of N. winogradskyi cells decreased less than those of N. europaea cells in all layers of the soil columns, probably because of heterotrophic growth of the nitrite-oxidizing bacteria on organic substrates excreted by the heterotrophic bacteria or because of nitrate reduction at reduced oxygen concentrations by the nitrite-oxidizing bacteria. Our conclusion was that the nitrifying bacteria were less competitive than the heterotrophic bacteria for ammonium in soil columns but that they survived as viable inactive cells. Inactive nitrifying bacteria may also be found in the rhizosphere of grassland plants, which is rich in organic carbon. They are possibly reactivated during periods of net mineralization.     

Penicillin-binding proteins inStreptococcus mitis

The penicillin-binding protein (PBP) profiles of penicillin-susceptible and-resistant clinical isolates ofStreptococcus mitis varied even with strains with similar minimal inhibitory concentrations (MICs).S. mitis NCTC 10712 was used as a DNA recipient to investigate PBP alterations which could occur as a result of spontaneous mutation and intra- and interspecific transfer of penicillin resistance genes.S. mitis NCTC 10712 possesses seven major PBPs ranging in molecular mass from 49–82 kDa. TwoS. mitis and twoStreptococcus pneumoniae penicillin-resistant clinical isolates were used as donors in transformation experiments withS. mitis NCTC 10712 (MIC 0.03 g/ml) as the recipient. Transformants with MICs greater than 1 g/ml were obtained with bothS. mitis andS. pneumoniae donor DNA. Depending on the source of the donor DNA and level of resistance achieved, transformants showed reduced penicillin-binding affinities of PBPs 2, 3, 4, 5, and 6. The most consistent PBP alteration associated with increasing resistance inS. mitis NCTC 10712 was seen with PBP 3 (74 kDa).     

Evidence for the Binding of Calmodulin to Endogenous B-50 (GAP-43) in Native Synaptosomal Plasma Membranes

The neuron-specific protein B-50 has been described as an atypical calmodulin (CaM) binding protein, because the purified protein has a higher affinity for CaM in the absence than in the presence of Ca2+. We have studied CaM binding to endogenous B-50 in native synaptosomal plasma membranes (SPM) and growth cone membranes in order to assess the physiological relevance of the binding. To detect B-50/CaM binding, we used the cross-linker disuccimidyl suberate (DSS) to form a covalent B-50/CaM complex, which is stable on SDS-PAGE. Upon addition of DSS, purified B-50 and calmodulin form a 70-kDa complex in the absence but not in the presence of Ca2+. This complex can be detected by protein staining and on Western blots using anti-B-50 and anti-CaM IgGs. DSS treatment of SPM or growth cone membranes with or without exogenous CaM results in the formation of a 70-kDa B-50/CAM complex detectable only in the absence of Ca2+ with both antibodies. Our results strongly suggest that the binding of CaM to endogenous B-50 in SPM and growth cone membranes is of physiological relevance. CaM binding to B-50 may be an important factor in regulating neurite outgrowth and/or neurotransmitter release.     

Transient-State Analysis of Metabolic Fluxes in Crabtree-Positive and Crabtree-Negative Yeasts

In bakers' yeast, an immediate alcoholic fermentation begins when a glucose pulse is added to glucose-limited, aerobically grown cells. The mechanism of this short-term Crabtree effect was investigated via a comparative enzymic analysis of eight yeast species. It was established that the fermentation rate of the organisms upon transition from glucose limitation to glucose excess is positively correlated with the level of pyruvate decarboxylase (EC 4.1.1.1). In the Crabtree-negative yeasts, the pyruvate decarboxylase activity was low and did not increase when excess glucose was added. In contrast, in the Crabtree-positive yeasts, the activity of this enzyme was on the average sixfold higher and increased after exposure to glucose excess. In Crabtree-negative species, relatively high activities of acetaldehyde dehydrogenases (EC 1.2.1.4 and EC 1.2.1.5) and acetyl coenzyme A synthetase (EC 6.2.1.1), in addition to low pyruvate decarboxylase activities, were present. Thus, in these yeasts, acetaldehyde can be effectively oxidized via a bypass that circumvents the reduction of acetaldehyde to ethanol. Growth rates of most Crabtree-positive yeasts did not increase upon transition from glucose limitation to glucose excess. In contrast, the Crabtree-negative yeasts exhibited enhanced rates of biomass production which in most cases could be ascribed to the intracellular accumulation of reserve carbohydrates. Generally, the glucose consumption rate after a glucose pulse was higher in the Crabtree-positive yeasts than in the Crabtree-negative yeasts. However, the respiratory capacities of steady-state cultures of Crabtree-positive yeasts were not significantly different from those of Crabtree-negative yeasts. Thus, a limited respiratory capacity is not the primary cause of the Crabtree effect in yeasts. Instead, the difference between Crabtree-positive and Crabtree-negative yeasts is attributed to differences in the kinetics of glucose uptake, synthesis of reserve carbohydrates, and pyruvate metabolism.     

Optimum conditions for efficient transformation ofStreptomyces venezuelae protoplasts

Summary Optimum conditions for protoplast regeneration and transformation ofStreptomyces venezuelae ETH 14630 have been established. Protoplasts from mycelium grown to the stationary phase and treated with lysozyme in P medium under mild conditions gave the best regeneration frequency. Transformation of protoplasts with naked DNA was very efficient using either polyethylene glycol of mol. wt. 4000 or 6000, at concentrations of 28.5% or 36% (w/v) respectively. About 10⁵ transformants/g DNA could be isolated using protoplasts derived from cells cultivated to the early exponential growth phase in LB medium containing 0.2%-0.6% glycine and subsequently treated at 30°–32°C with 20 mg lysozyme/ml in P medium for 30 min. Selection of the transformants occurred on MRYE plates containing less than 10⁵ regenerating protoplasts per plate. Higher protoplast densities considerably decreased the regeneration frequency of the transformants.     

H-nmr study on the preferred conformations of chiral pyridine-dinucleotide models

The synthesis of four chiral NAD⁺ models 1 and their 1,4-dihydro analogs 2 is described. From the temperature dependence of the ¹H-nmr spectra it is concluded that for these compounds two preferred conformations I and II, differing slightly in energy, exist. Both conformations are “folded” with the more or less parallel p-anisyl and pyridine groups mutually gauche, but in I the pyridine group is rotated by about 180° as compared with II, thus leading to a conspicuous difference in orientation of the substituent Z (NH₂CO, C₆H₅NHSO₂, (CH₂)₄NSO₂, or (C₄H₈ON)SO₂) in the pyridine ring toward the anisyl group. The most stable conformation (I) has Z closest to the center of the p-anisyl group. In 360-MHz spectra of the dihydropyridines at low temperature (−10°C), slow interconversion of I and II leads to the observation of an XY pattern for the C-4 methylene protons of the 1,4-dihydropyridine system. The anisochronity in this methylene group is caused mainly by the anisotropy of the neighboring p-anisyl group.     

Interspecific hybridization between Penicillium chrysogenum and Penicillium cyaneo-fulvum following protoplast fusion

Summary Slow-growing interspecific heterokaryons were isolated on minimal medium following the induced fusion of protoplasts from auxotrophic mutants of Penicillium chrysogenum and Penicillium cyaneo-fulvum. After 5–7 days cultivation the heterokaryons produced vigorously growing sectors which on transfer gave genetically stable colonies. Cultivation of these colonies on a complete medium supplemented with p-fluorophenylalanine or benomyl broke down this stability and several different prototrophic and auxotrophic colony types were isolated. Many of these behaved as diploids or aneuploids showing sectoring either spontaneously, or in the presence of an haploidizing agent. Some of the latter isolates were recombinants for parental spore colour and auxotrophic markers.     

Orientation sensitivity of the visual movement detection system activating the landing response of the blowflies,Calliphora, and Phaenicia: A behavioural investigation

The orientation sensitivity of the visual movement detection system relative to the axes of the eye was investigated for the landing response by changing the direction of movement of a periodic striped pattern (unidirectional movement) and a two-stripe pattern consiting of two stripes moving apart (bidirectional movement). In the momocular, equatorial regions of the eye progressive motion proves to be most effective, whereas in the frontal (equational), binocular region descendive motion is most effective in eliciting the landing response, probably caused by binocular interactions. A strong enhancement of the response is induced by stimulation in the binocular region of the two eyes. The orientation of elementary movement detectors relative to the axes of the ommatidial array is discussed. The findings are summarized in a functional model of the landing response.     

Condition‐dependence and sexual ornamentation: Effects of immune challenges on a highly sexually dimorphic grasshopper

Sexual ornaments contribute substantially to phenotypic diversity and it is particularly relevant to understand their evolution. Ornaments can assume the function of signals‐of‐quality that the choosy sex uses to evaluate potential mating partners. Often there are no obvious direct benefits and investment into mate choice is primarily rewarded by beneficial alleles that are inherited to the offspring. Inter‐sexual communication via sexual ornaments requires honesty of the sexual signal, yet the question of what maintains honesty remains only partially solved. One solution is that honesty is maintained by trait expression being dependent on individual condition, since condition‐dependent trait expression offers an effectively inexhaustible source of genetic variability. Here we test in the highly sexually dimorphic club‐legged grasshopper Gomphocerus sibiricus if putative sexual ornaments, in particular the striking front‐leg clubs, are more strongly affected by a lipopolysaccharide (LPS) immune challenge than putatively not sexually selected traits. Our results show overall little condition‐dependent expression of morphological and song traits, with sexually selected traits exhibiting effects comparable to nonsexually selected traits (with the possible exception of stridulatory file length and syllable‐to‐pause ratio in advertisement songs). Interestingly, field observations of individuals of lethally parasitized individuals suggest that a very strong environmental challenge can specifically affect the expression of the front‐leg clubs. The presence of 1% of males in natural populations with missing or heavily deformed clubs plus 5% with minor club deformations furthermore indicate that there are risks associated with club development during final ecdysis and this might act as a filter against deleterious alleles.