The size and position of heterologous insertions in a silent locus differentially affect pilin recombination in Neisseria gonorrhoeae

Gonococcal pilus antigenic and phase variation result from unidirectional, RecA-dependent recombination of DNA sequences from a silent pilin copy ( pilS  ) into the expressed pilin gene ( pilE  ). To develop a quantitative assay for pilin gene recombination that is independent of phase variation, a promoterless cat gene was inserted into pilS , and recombination of ' cat into pilE was detected by selection of chloramphenicol-resistant (Cm^R) variants expressing ' cat from the pilin promoter. Although RecA-dependent Cm^R variants occurred, none were generated by the simple transfer of ' cat into pilE . Instead, each Cm^R variant contained a new pilin locus that was a hybrid of sequences from the pilE and the pilS1 ::' cat loci in addition to the two starting loci. Therefore, this system could not be used to quantify antigenic variation. However, combined studies of these hybrid loci and of recombination products generated during additional pilS mutational analyses demonstrated that both the size and position of an insertion in pilS differentially affect pilin recombination. Also, the hybrid loci appear to be intermediates of antigenic variation. This enabled the creation of molecular models for the recombination reactions that result in pilin antigenic variation.     

Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations.

We have generated a mouse carrying the human G551D mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a one-step gene targeting procedure. These mutant mice show cystic fibrosis pathology but have a reduced risk of fatal intestinal blockage compared with 'null' mutants, in keeping with the reduced incidence of meconium ileus in G551D patients. The G551D mutant mice show greatly reduced CFTR-related chloride transport, displaying activity intermediate between that of cftr(mlUNC) replacement ('null') and cftr(mlHGU) insertional (residual activity) mutants and equivalent to approximately 4% of wild-type CFTR activity. The long-term survival of these animals should provide an excellent model with which to study cystic fibrosis, and they illustrate the value of mouse models carrying relevant mutations for examining genotype-phenotype correlations.     

ISOLATION AND CHARACTERIZATION OF GOLGI FROM COCCOLITHUS PELAGICUS (PRYMNESIOPHYCEAE)1

Cells of the marine alga Coccolithus pelagicus (Wal-lich)J. Schiller grown in axenic cultures were homogenized and fractionated. The distribution of organelle markers was assessed enzymatically after centrifugation through zonal, density, and flotation gradients made with sucrose, sorbitol, or Percoll. Mitochondria (1.19 g·cm^-3) and chloroplasts (1.15 g·cm^-3) were recovered in sucrose gradients at densities similar to those observed for higher plants and most algae. The position of endoplasmic reticulum and plasma membrane in the gradients was monitored by NADPH cytochrome c reductase and vanadate-sensitive Mg²⁺-ATPase, respectively. Higher plant Golgi markers, latent undine diphosphatase (UDPase) and glucan synthase I, were colocalized at a density range including two peaks of activity at 1.13–1.15 g·cm^-3. Bound calcium was associated with high density (1.15 g·cm^-3) membranes. Ca²⁺-stimulated ATPase was found at high levels on membranes that did not coisolate with the latent UDPase-containing membranes. The Ca²⁺-stimulated ATPase, a possible participant during calcification, was associated with a chloroplast-enriched fraction in all the organelle separation systems. However, about 30% of the total activity was separated from both the chloroplasts and Golgi on 0–70% Percoll gradients containing 0.4 M sucrose. The possible relationship of the Golgi and the high-density organelle exhibiting Ca²⁺-stimulated ATPase to coccolithogenesis and the process of calcification and crystal formation is discussed.     

Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes

Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione.     

Usurpation of a Crowned Lapwing Vanellus coronatus nest by African Wattled Lapwings V. senegalensis

I report an instance of usurpation of a Crowned Lapwing Vanellus coronatus nest by a pair of African Wattled Lapwings Vanellus senegalensis. The nest, which originally contained a single Crowned Lapwing egg, eventually contained an additional three Wattled Lapwing eggs, before it was predated. Although parents of both species were observed in the vicinity of the nest, video monitoring showed that the Wattled Lapwings incubated all four eggs. Detection of embryonic heart rate suggested that all the eggs were probably viable up until the nest was destroyed. Nest usurpation in shorebirds is rare, and given the easily accommodated nest requirements and minimal costs of nest construction in these species, is difficult to explain. I speculate that the occurrence described here may represent a case of mistaken identity by the female Wattled Lapwing when choosing a nest scrape, since there seems to have been little benefit to the usurping species of taking over an already occupied nest.     

Identification and analysis of integrons and cassette arrays in bacterial genomes

Integrons recombine gene arrays and favor the spread of antibiotic resistance. Their broader roles in bacterial adaptation remain mysterious, partly due to lack of computational tools. We made a program – IntegronFinder – to identify integrons with high accuracy and sensitivity. IntegronFinder is available as a standalone program and as a web application. It searches for attC sites using covariance models, for integron-integrases using HMM profiles, and for other features (promoters, attI site) using pattern matching. We searched for integrons, integron-integrases lacking attC sites, and clusters of attC sites lacking a neighboring integron-integrase in bacterial genomes. All these elements are especially frequent in genomes of intermediate size. They are missing in some key phyla, such as α-Proteobacteria, which might reflect selection against cell lineages that acquire integrons. The similarity between attC sites is proportional to the number of cassettes in the integron, and is particularly low in clusters of attC sites lacking integron-integrases. The latter are unexpectedly abundant in genomes lacking integron-integrases or their remains, and have a large novel pool of cassettes lacking homologs in the databases. They might represent an evolutionary step between the acquisition of genes within integrons and their stabilization in the new genome.     

Extending bioacoustic monitoring of birds aloft through flight call localization with a three‐dimensional microphone array

Bioacoustic localization of bird vocalizations provides unattended observations of the location of calling individuals in many field applications. While this technique has been successful in monitoring terrestrial distributions of calling birds, no published study has applied these methods to migrating birds in flight. The value of nocturnal flight call recordings can increase with the addition of three‐dimensional position retrievals, which can be achieved with adjustments to existing localization techniques. Using the time difference of arrival method, we have developed a proof‐of‐concept acoustic microphone array that allows the three‐dimensional positioning of calls within the airspace. Our array consists of six microphones, mounted in pairs at the top and bottom of three 10‐m poles, arranged in an equilateral triangle with sides of 20 m. The microphone array was designed using readily available components and costs less than $2,000 USD to build and deploy. We validate this technique using a kite‐lofted GPS and speaker package, and obtain 60.1% of vertical retrievals within the accuracy of the GPS measurements (±5 m) and 80.4% of vertical retrievals within ±10 m. The mean Euclidian distance between the acoustic retrievals of flight calls and the GPS truth was 9.6 m. Identification and localization of nocturnal flight calls have the potential to provide species‐specific spatial characterizations of bird migration within the airspace. Even with the inexpensive equipment used in this trial, low‐altitude applications such as surveillance around wind farms or oil platforms can benefit from the three‐dimensional retrievals provided by this technique.     

Linkage of cystic fibrosis to two tightly linked DNA markers: Joint report from a collaborative study

A collaborative study involving seven research groups provided an opportunity to investigate the linkage relationships between cystic fibrosis and two DNA marker loci, MET and pJ3.11 (D7S8), on an extended sample of 211 tested families. The maximum lod scores, recombination estimates, and confidence upper bounds (in parentheses) were 91.0 at theta = .004 (.012) for CF and MET, 71.3 at theta = .003 (.011) for CF and D7S8, and 69.3 at theta = .018 (.036) for MET and D7S8. Three-locus analyses yielded best support for the order MET-CF-D7S8, with odds against the alternate orders CF-MET-D7S8 and CF-D7S8-MET of 9:1 and 161:1, respectively. However, the number of observed recombinants was small and only one of the recombinants was jointly informative for all three markers. Significant allelic association was found between CF and both MET and D7S8. Weaker association between the latter two loci is consistent with the order MET-CF-D7S8.