Characterization ofH-2 congenic strains using DNA markers

Congenic mouse strains are widely used in mapping traits to specific loci or short chromosomal regions. The precision of the mapping depends on the information available about the length of the differential segment—the segment introduced from the donor into the background strain. Until recently, very few markers flanking the differential locus were known and consequently the length of the foreign segment could only be determined imprecisely. Now, in an attempt to construct a map of the mouse chromosome 17, we have produced a set of DNA markers distributed along the chromosome. These markers provide a new opportunity to measure the length of the differential segment of the congenic strains and thus increase their usefulness for gene mapping. Here we examined the DNA of 96H-2 congenic strains using 30 DNA markers; of these, the most proximal is located roughly 1.5 centiMorgans (cM) from the centromere and the most distal is about 20 cM telomeric from theH-2 complex (the complex itself being some 20 cM from the centromere). The mapping depends on polymorphism among the input strains and can therefore establish only the minimal length of the differential segment. This point is emphasized by the fact that the average observed length of the differential segment is only about one half of the expected values. Offprint requests to: V. Vincek.     

Molecular polymorphism and phenotypic variation in Aspergillus carbonarius

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.Abbreviations kb kilobase pair - mtDNA mitochondrial DNA - RAPD random amplified polymorphic DNA - rDNA ribosomal RNA gene cluster - RFLP restriction fragment length polymorphisms     

Translation termination in human mitochondrial ribosomes

Mitochondria are ubiquitous and essential organelles for all nucleated cells of higher eukaryotes. They contain their own genome [mtDNA (mitochondrial DNA)], and this autosomally replicating extranuclear DNA encodes a complement of genes whose products are required to couple oxidative phosphorylation. Sequencing of this human mtDNA more than 20?years ago revealed unusual features that included a modified codon usage. Specific deviations from the standard genetic code include recoding of the conventional UGA stop to tryptophan, and, strikingly, the apparent recoding of two arginine triplets (AGA and AGG) to termination signals. This latter reassignment was made because of the absence of cognate mtDNA-encoded tRNAs, and a lack of tRNAs imported from the cytosol. Each of these codons only occurs once and, in both cases, at the very end of an open reading frame. The presence of both AGA and AGG is rarely found in other mammals, and the molecular mechanism that has driven the change from encoding arginine to dictating a translational stop has posed a challenging conundrum. Mitochondria from the majority of other organisms studied use only UAA and UAG, leaving the intriguing question of why human organelles appear to have added the complication of a further two stop codons, AGA and AGG, or have they? In the present review, we report recent data to show that mammalian mitochondria can utilize a -1 frameshift such that only the standard UAA and UAG stop codons are required to terminate the synthesis of all 13 polypeptides.     

The effect of drought hardening and chilling on ABA content in xylem sap and ABA - delivery rate from root of tomato plant

This paper is a continuation of our studies related to the response of two tomato cultivars: Robin and New Yorker to chilling: the later is more tolerant to chilling than the former one (Starck et al. 1994). The concentration of ABA in the xylem sap and ABA delivery rate (calculated as the amount of ABA exuded in 2h from the cut stump, following shoot removal) were estimated by ELISA. The relative water content (RWC) of the leaf blades and stomatal resistance (RS) were also measured. Tomato plants were grown in a greenhouse, under noncontrolled conditions. Before chilling some of the plants were drought hardened for 10 days (H). As an consequence of water deficit only New Yorker growth slightly decreased. Plants were chilled to 2–5 °C during three consecutive, 16-h nights, preceded by warm days, which caused a decrease in the RWC of leaf blades. Chilling did not decreased leaf blade hydration significantly, but drastically increased the concentration of ABA in the xylem sap in more chilling tolerant cv. New Yorker only. The delivery rate of ABA was markedly enhanced in both cultivars, but much more in New Yorker. Drought hardening increased ABA delivery rate in cv. Robin only, especially after chilling. The lack of correlation between changes in the RWC of leaf blades after low temperature treatment and the concentration of ABA in the xylem sap as well as its delivery rate suggest, that in both tomato cultivars chilling increased ABA level directly, not as an secondery effect of temperature-induced water deficit.     

Biodegradation kinetics of 2,4-D by bacterial strains isolated from soil

The aim of the study was to characterize the 2,4-dichlorophenoxyacetic acid (2,4-D) degradative potential of three bacterial strains identified by MIDI-FAME profiling as Burkholderia cepacia (DS-1), Pseudomonas sp. (DS-2) and Sphingomonas paucimobilis (DS-3) isolated from soil with herbicide treatment history. All strains were capable of using herbicide as the only source of carbon and energy when grown in mineral salt medium (MSM) containing 2,4-D (50 mg/l). Over a 10 day incubation period, 69%, 73% and 54% of the initial dose of 2,4-D were degraded by strains DS-1, DS-2 and DS-3, respectively. Analysis of 2,4-dichlorophenol (2,4-DCP) concentration, the main metabolite of 2,4-D degradation, revealed that strains DS-1 and DS-2 may also have the potential to metabolize this compound. The percentage of 2,4-DCP removal was 67% and 77% in relation to maximum values of 9.5 and 9.2 mg/l determined after 4 and 2 days for MSM+DS-1 and MSM+DS-2, respectively. The degradation kinetics of 2,4-D (50 mg/kg) in sterile soil (SS) showed different potential of tested strains to degrade 2,4-D. The times within which the initial 2,4-D concentration was reduced by 50% (DT₅₀) were 6.3, 5.0 and 9.4 days for SS+DS-1, SS+DS-2 and SS+DS-3, respectively.     

DNA Physical Properties and Nucleosome Positions Are Major Determinants of HIV-1 Integrase Selectivity

Retroviral integrases (INs) catalyse the integration of the reverse transcribed viral DNA into the host cell genome. This process is selective, and chromatin has been proposed to be a major factor regulating this step in the viral life cycle. However, the precise underlying mechanisms are still under investigation. We have developed a new in vitro integration assay using physiologically-relevant, reconstituted genomic acceptor chromatin and high-throughput determination of nucleosome positions and integration sites, in parallel. A quantitative analysis of the resulting data reveals a chromatin-dependent redistribution of the integration sites and establishes a link between integration sites and nucleosome positions. The co-activator LEDGF/p75 enhanced integration but did not modify the integration sites under these conditions. We also conducted an in cellulo genome-wide comparative study of nucleosome positions and human immunodeficiency virus type-1 (HIV-1) integration sites identified experimentally in vivo. These studies confirm a preferential integration in nucleosome-covered regions. Using a DNA mechanical energy model, we show that the physical properties of DNA probed by IN binding are important in determining IN selectivity. These novel in vitro and in vivo approaches confirm that IN has a preference for integration into a nucleosome, and suggest the existence of two levels of IN selectivity. The first depends on the physical properties of the target DNA and notably, the energy required to fit DNA into the IN catalytic pocket. The second depends on the DNA deformation associated with DNA wrapping around a nucleosome. Taken together, these results indicate that HIV-1 IN is a shape-readout DNA binding protein.     

The phylogenetic relationship of tetrapod, coelacanth, and lungfish revealed by the sequences of forty-four nuclear genes

The origin of tetrapods is a major outstanding issue in vertebrate phylogeny. Each of the three possible principal hypotheses (coelacanth, lungfish, or neither being the sister group of tetrapods) has found support in different sets of data. In an attempt to resolve the controversy, sequences of 44 nuclear genes encoding amino acid residues at 10,404 positions were obtained and analyzed. However, this large set of sequences did not support conclusively one of the three hypotheses. Apparently, the coelacanth, lungfish, and tetrapod lineages diverged within such a short time interval that at this level of analysis, their relationships appear to be an irresolvable trichotomy.     

Mycoflora and mycotoxins in Brazilian black pepper,white pepper and Brazil nuts

A wide range of field and storage fungi were isolated from black pepper, white pepper and Brazil nut kernels from Amazonia. A total of 42 species were isolated from both peppers. Aspergillus flavus and A. niger were isolated more frequently from black than from white pepper. Other potential mycotoxigenic species isolated included: A. ochraceus, A. tamarii, A. versicolor, Emericella nidulans and Chaetomium globosum, Penicillium brevicompactum, P. citrinum, P. islandicum and P. glabrum. Species isolated from pepper for the first time were Acrogenospora sphaerocephala, Cylindrocarpon lichenicola, Lacellinopsis sacchari, Microascus cinereus, Petriella setifera and Sporormiella minima. Seventeen species were isolated from Brazil nut kernels. A. flavus was the dominant species followed by A. niger. P. citrinum and P. glabrum were the only penicillia isolated. Species isolated for the first time included Acremonium curvulum, Cunninghamella elegans, Exophiala sp., Fusarium oxysporum, Pseudoallescheria boydii, Rhizopusoryzae, Scopulariopsis sp., Thielavia terricola and Trichoderma citrinoviride. Considerably more metabolites were detected from black than white pepper in qualitative analyses. Chaetocin, penitrem A, and xanthocillin were identified only from black pepper, and tenuazonic acid was identified from both black and white pepper. Aflatoxin G₂, chaetoglobosin C, and spinulosin were identified from poor quality brazil nuts. Aflatoxin B₁ and B₂ were also only detected in poor quality brazil nuts at concentrations of 27.1 g kg^–1 and 2.1 g kg^–1 respectively (total 29.2 g kg^–1).This revised version was published online in October 2005 with corrections to the Cover Date.     

FAME profiles in Pseudomonas vesicularis during catechol and phenol degradation in the presence of glucose as an additional carbon source.

The aim of this study was to evaluate the impact of catechol and phenol added to culture media separately and with glucose as an additional, easily-degradable carbon source on fatty acid methyl ester (FAME) composition in Pseudomonas vesicularis. Simultaneously, the degradation rates of aromatic substrates used were investigated in single and binary substrate systems. Both catechol and phenol treatments caused changes in the distribution of tested groups of fatty acids. The most noticeable changes included an increase in degree of fatty acid saturation, the appearance of branched and disappearance of hydroxy fatty acids as compared to the control sample with glucose. Under catechol or phenol treatment sat/unsat ratio showed the values of 8.63 and 11.38, respectively, whereas in control cells it reached the value of 2.66. The high level of saturation comes from the high content of cyclopropane fatty acids in bacteria under exposure to aromatic substrates, regardless of the presence of glucose. In these treatments their content was more than 3-fold higher compared to the control. It has been demonstrated that glucose supplementation of culture media containing single aromatic substrate extended the degradation rates of catechol and phenol by P. vesicularis, caused an increase in number of cells but did not significantly change the fatty acid profiles in comparison with bacteria growing on catechol and phenol added to the media individually.