Recognition of single-stranded DNA by nuclease P1: High resolution crystal structures of complexes with substrate analogs

The reaction mechanism of nuclease P1 from Penicillium citrinum has been investigated using single-stranded dithiophosphorylated di-, tetra-, and hexanucleotides as substrate analogs. The complexes crystallize in tetragonal and orthorhombic space groups and have been solved by molecular replacement. The high resolution structures give a clear picture of base recognition by P1 nuclease at its two nucleotide-binding sites, especially the 1.8 Å structure of a P1-tetranucleotide complex which can be considered a P1-product complex. The observed binding modes are in agreement with a catalytic mechanism where the two closely spaced zinc ions activate the attacking water while the third, more exposed zinc ion stabilizes the leaving 03' oxyanion. Stacking as well as hydrogen bonding interactions with the base 5' to the cleaved phosphodiester bond are important elements of substrate binding and recognition. Modelling of a productive P1-substrate complex based on the solved structures suggests steric hindrance as the likely reason for the resistance of Rp-phosphorothioates and phosphorodithioates. Differences with the highly homologous nuclease S1 from Aspargillus oryzae are discussed. Proteins 32:414–424, 1998. © 1998 Wiley-Liss, Inc.     

Anesthesia of wild red howler monkeys (Alouatta seniculus) with medetomidine/ketamine and reversal by atipamezole

Wild red howler monkeys (Alouatta seniculus) were translocated during the flooding of the forest at a hydroelectric dam site in French Guiana. For a variety of minor clinical procedures, 96 monkeys were anesthetized with various intramuscular injections of combinations of medetomidine and ketamine. The howler population was composed of healthy animals (42 males and 54 females) of various ages. Medetomidine (150 μg/kg) associated with ketamine (4 mg/kg) gave the best results and was used on 63 animals. The injection rapidly resulted in complete immobilization with good to excellent myorelaxation. The induction stage was quiet, with absence of both corneal and pedal withdrawal reflexes in 57 animals after 2.9 ± 1.4 min. Six animals required an additional injection. Rectal temperature and respiratory and heart rates decreased during anesthesia, whereas relative oxyhemoglobin saturation increased. One death occurred during anesthesia. One abortion and one death also occurred the day following anesthesia but were more probably a result of capture stress. Atipamezole given i.m. at a dose of five times the medetomidine dose 38.4 ± 8.0 min after the anesthetic injection led to standing recovery in 7.1 ± 4.5 min. Spontaneous recovery occurred in 17 animals before the atipamezole injection after an average of 30.6 ± 9.6 min. Total recovery time was shorter in young animals. Medetomidine/ketamine induced good myorelaxation and provided considerably shortened immobilization duration, which are two notable advantages for field studies. We recommend this association for short procedures including minor surgery in red howler monkeys. Am. J. Primatol. 45:399–410, 1998. © 1998 Wiley-Liss, Inc.     

CBP70, a glycosylated nuclear lectin

Some years ago, a lectin designated CBP70 that recognized glucose (Glc) but had a stronger affinity for N-acetylglucosamine (GlcNAc), was first isolated from HL60 cell nuclei. Recently, a cytoplasmic form of this lectin was described, and one 82 kDa nuclear ligand was characterized for the nuclear CBP70. In the present study, the use of Pronase digestion and the trifluoromethanesulphonic acid (TFMS) procedure strongly suggest that the nuclear and the cytoplasmic CBP70 have a same 23 kDa polypeptide backbone and, consequently, could be the same protein. In order to know the protein better and to obtain the best recombinant possible in the future, the post-translational modification of the nuclear and cytoplasmic CBP70 was analyzed in terms of glycosylation. Severals lines of evidence indicate that both forms of CBP70 are N- and O-glycosylated. Surprisingly, this glycosylation pattern differs between the two forms, as revealed by β-elimination, hydrazinolysis, peptide-N-glycosydase F (PNGase F), and TFMS reactions. The two preparations were analyzed by affinity chromatography on immobilized lectins [Ricinus communis-I agglutinin (RCA-I), Arachis hypogaea agglutinin (PNA), Galanthus nivalis agglutinin (GNA), and wheat germ agglutinin (WGA)] and by lectin-blotting analysis [Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Lotus tetragonolobus (Lotus), succinylated-WGA, and Psathyrella velutina agglutinin (PVA)]. Both forms of CBP70 have the following sugar moities: terminal βGal residues, Galβ1–3 GalNAc, Man α1–3 Man, sialic acid α2–6 linked to Gal or GalNAc; and sialic acid α2–3 linked to Gal. However, only nuclear CBP70 have terminal GlcNAc and α-L-fucose residues. All these data are consistent with the fact that different glycosylation pattern found for each form of CBP70 might act as a complementary signal for cellular targeting. J. Cell. Biochem. 66:370–385, 1997. © 1997 Wiley-Liss, Inc.     

Factors that influence bidirectional long-tract homozygosis due to double-strand break repair in Candida albicans

Genomic rearrangements have been associated with the acquisition of adaptive phenotypes, allowing organisms to efficiently generate new favorable genetic combinations. The diploid genome of Candida albicans is highly plastic, displaying numerous genomic rearrangements that are often the by-product of the repair of DNA breaks. For example, DNA double-strand breaks (DSB) repair using homologous-recombination pathways are a major source of loss-of-heterozygosity (LOH), observed ubiquitously in both clinical and laboratory strains of C. albicans. Mechanisms such as break-induced replication (BIR) or mitotic crossover (MCO) can result in long tracts of LOH, spanning hundreds of kilobases until the telomere. Analysis of I-SceI-induced BIR/MCO tracts in C. albicans revealed that the homozygosis tracts can ascend several kilobases toward the centromere, displaying homozygosis from the break site toward the centromere. We sought to investigate the molecular mechanisms that could contribute to this phenotype by characterizing a series of C. albicans DNA repair mutants, including pol32^-/-, msh2^-/-, mph1^-/-, and mus81^-/-. The impact of deleting these genes on genome stability revealed functional differences between Saccharomyces cerevisiae (a model DNA repair organism) and C. albicans. In addition, we demonstrated that ascending LOH tracts toward the centromere are associated with intrinsic features of BIR and potentially involve the mismatch repair pathway which acts upon natural heterozygous positions. Overall, this mechanistic approach to study LOH deepens our limited characterization of DNA repair pathways in C. albicans and brings forth the notion that centromere proximal alleles from DNA break sites are not guarded from undergoing LOH.     

Characterization of the fungal flora of dolo,a traditional fermented beverage of Burkina Faso,using MALDI-TOF mass spectrometry

A cross-sectional study was conducted in Bobo-Dioulasso, Burkina Faso, to identify the yeast diversity associated with the manufacture of dolo, a traditional fermented beverage of Burkina Faso. From sixty specimens spread onto chromogenic medium plates, sixty-two strains were isolated then identified using MALDI-TOF analysis. Seven yeast species were identified, Saccharomyces cerevisiae (39%) followed by Pichia manshurica (18%) being the most frequent. Forty-three percent of the samples contained Candida species, notably Candida albicans. In conclusion, the combined use of a chromogenic medium and MALDI-TOF analysis reveals a higher diversity in yeast species present in the dolo than previously thought.     

Dmrt1 polymorphism and sex‐chromosome differentiation in Rana temporaria

Sex‐determination mechanisms vary both within and among populations of common frogs, opening opportunities to investigate the molecular pathways and ultimate causes shaping their evolution. We investigated the association between sex‐chromosome differentiation (as assayed from microsatellites) and polymorphism at the candidate sex‐determining gene Dmrt1 in two Alpine populations. Both populations harboured a diversity of X‐linked and Y‐linked Dmrt1 haplotypes. Some males had fixed male‐specific alleles at all markers (“differentiated” Y chromosomes), others only at Dmrt1 (“proto‐” Y chromosomes), while still others were genetically indistinguishable from females (undifferentiated X chromosomes). Besides these XX males, we also found rare XY females. The several Dmrt1 Y haplotypes differed in the probability of association with a differentiated Y chromosome, which we interpret as a result of differences in the masculinizing effects of alleles at the sex‐determining locus. From our results, the polymorphism in sex‐chromosome differentiation and its association with Dmrt1, previously inferred from Swedish populations, are not just idiosyncratic features of peripheral populations, but also characterize highly diverged populations in the central range. This implies that an apparently unstable pattern has been maintained over long evolutionary times.     

Protein–nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR

DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein–DNA and protein–ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein–DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy. Interactions are established by DNP-enhanced ³¹P–¹³C polarization-transfer experiments followed by the recording of a 2D ¹³C–¹³C correlation experiment. The NMR spectra were obtained in less than 2 days and allowed the identification of residues of the motor protein involved in nucleotide binding.