Phylogenetic screening of a bacterial,metagenomic library using homing endonuclease restriction and marker insertion

Metagenomics provides access to the uncultured majority of the microbial world. The approaches employed in this field have, however, had limited success in linking functional genes to the taxonomic or phylogenetic origin of the organism they belong to. Here we present an efficient strategy to recover environmental DNA fragments that contain phylogenetic marker genes from metagenomic libraries. Our method involves the cleavage of 23S ribsosmal RNA (rRNA) genes within pooled library clones by the homing endonuclease I-CeuI followed by the insertion and selection of an antibiotic resistance cassette. This approach was applied to screen a library of 6500 fosmid clones derived from the microbial community associated with the sponge Cymbastela concentrica. Several fosmid clones were recovered after the screen and detailed phylogenetic and taxonomic assignment based on the rRNA gene showed that they belong to previously unknown organisms. In addition, compositional features of these fosmid clones were used to classify and taxonomically assign a dataset of environmental shotgun sequences. Our approach represents a valuable tool for the analysis of rapidly increasing, environmental DNA sequencing information.     

Plant communities of a Central Namib inselberg landscape

Abstract. Three granite inselbergs and six dolerite dykes and their surroundings were investigated in the Central Namib, at the interface between the Namib Desert and Nama Karoo biomes. The main objectives of this study included a phytoso‐ciological interpretation of the described plant communities, explanation of the correlation of the communities with environmental variables and quantification of the relative contribution of different types of variables to structuring plant communities. Nine grassland and shrubland plant communities were recognized, largely organized according to general habitat, elevation, size of inselberg and geology. Soil properties, often thought to play an important role in arid environments, showed no clear patterns in the level of analyses used in this study. Other environmental parameters of importance in arid mountain habitats, such as slope aspect and angle, also played a minor role. The main implications of the study are: 1. Central Namib inselbergs, particularly granite domes, harbour diverse plant communities, often with species from neighbouring higher rainfall areas, and are thus of high conservation value. 2. The poor contribution of environmental variables in this study, which are conventionally used in field studies of plant community – environment relationships, may demand a critical review of additional parameters to be included when analysing plant community – environment relations in arid environments. In particular between‐season variation, phytogeographic aspects and the heterogeneity of microhabitats, often contained within a plant community, need to be taken into account.     

Floral expression of a gene encoding an E2-relatedubiquitin-conjugating protein from Arabidopsis thaliana

An Arabidopsis thaliana gene (UBC6) encoding a homologue to ubiquitin-conjugating enzymes has been isolated which is capable of encoding a protein of 183 amino acids of ca. 21 kDa. Northern analysis indicates that the gene is expressed in flowers, seeds and, to a somewhat lesser extent, in 10-day seedlings but not in mature leaves, callus and pre-flowering plants. This pattern of expression is confirmed using transgenic Arabidopsis plants containing a UBC6 promoter-GUS gene fusion construct. These plants displey GUS activity in mature anthers prior to dehiscence, in developing embryos, sepals and the style after pollination.     

Determination of 4-hydroxyproline in collagen by gas chromatography/mass spectrometry

Derivatization of 4-hydroxyproline (Hyp) in collagen using trifluoroacetylation and methanol esterification produces two derivatives when analyzed by gas chromatography/mass spectrometry (GC/MS). The diacyl derivative N,O-bis(trifluoroacetyl)-4-hydroxy-L-proline methyl ester (N,O-TFA-Hyp) formed in this manner has a shorter retention time and different fragmentation pattern by GC/MS as compared to the slower eluting monoacetylated species N-trifluoroacetyl-4-hydroxy-L-proline methyl ester (N-TFA-Hyp). By selected ion monitoring of the appropriate ions of either N,O-TFA-Hyp (m/z 164, 278) or N-TFA-Hyp (m/z 164, 182) efficient quantitation of Hyp in collagen is possible within the broad range of 5-1000 ng with a lower limit of detection of 0.5 ng per injection. Measurement of 18O2 incorporation into collagen is possible by selected ion monitoring of the m/z 182 ion formed only from the monoacetylated derivative, N-TFA-Hyp, produced by methanol solvolysis of the N,O-TFA-Hyp derivative, as proposed herein.     

The hairpin ribozyme substrate binding-domain: a highly constrained D-shaped conformation

The two domains of the hairpin ribozyme-substrate complex, usually depicted as straight structural elements, must interact with one another in order to form an active conformation. Little is known about the internal geometry of the individual domains in an active docked complex. Using various crosslinking and structural approaches in conjunction with molecular modeling (constraint-satisfaction program MC-SYM), we have investigated the conformation of the substrate-binding domain in the context of the active docked ribozyme-substrate complex. The model generated by MC-SYM showed that the domain is not straight but adopts a bent conformation (D-shaped) in the docked state of the ribozyme, indicating that the two helices bounding the internal loop are closer than was previously assumed. This arrangement rationalizes the observed ability of hairpin ribozymes with a circularized substrate-binding strand to cleave a circular substrate, and provides essential information concerning the organization of the substrate in the active conformation. The internal geometry of the substrate-binding strand places G8 of the substrate-binding strand near the cleavage site, which has allowed us to predict the crucial role played by this nucleotide in the reaction chemistry.     

Environmental and Seasonal Factors Affecting the Frost-induced Stage of Cold Acclimation in Cornus stolonifera Michx

Stem tissues of red-osier dogwood (Cornus stolonifera Michx.) acclimated from −3 C to −40 or −50 C in 8 to 10 weeks under a short photoperiod (9 hours) and controlled temperature conditions. During the summer months plants did not acclimate as well as at other times. The sequence of day/night temperature regimes which induced maximum acclimation was 20/15 C for 5 to 6 weeks; 15/5 C for 2 to 3 weeks; 15/5 C plus 1 hour of frost per day for 1 week. The duration of exposure to each temperature regime influenced the rate and intensity of frost-induced acclimation. Less than 5 weeks of warm temperature preconditioning at 20/15 C reduced subsequent frost-induced acclimation. The inductive influence of frost on cold acclimation was additive over 5 days of repeated exposure, but its effects after the first exposure(s) were not immediate—requiring 1 to 4 days of 15/5 C following the frost treatments for the expression of the frost-induced acclimation to be manifest. There was a 75% increase in rRNA following 3 days of frost exposure and plants in an O₂-free atmosphere during frost exposure failed to acclimate. The results suggest that seasonal acclimation behavior was due to endogenous rhythms rather than developmental stage, and that the frost-induced phase of acclimation involves aerobic metabolic processes.     

Interaction between the A2 and A19 amino acid residues is of critical importance for high biological activity in insulin: [19-leucine-A]insulin

The replacement of tyrosine at position A19 by leucine in the insulin molecule led to an analogue, [19-leucine-A]insulin [( Leu19-A]insulin), displaying insignificant receptor binding affinity and in vitro biological activity less than 0.1 and 0.05%, respectively, compared to the natural hormone. This analogue along with the previously reported [2-glycine-A]-, [2-alanine-A]-, and [2-norleucine-A]insulins is the least potent insulin analogue we have examined. Circular dichroic studies showed that all these analogues are monomeric at concentrations at which insulin is primarily dimeric. We conclude that an aromatic ring at position A19 and the presence of the side chain of isoleucine at position A2 are each of critical importance for high biological activity in insulin. It appears that the van der Waals interaction between the side chain of isoleucine A2 and tyrosine A19, present in crystalline insulin, is among the most important determinants for high biological activity in insulin.