A macromolecule which gives rise to thyrotropin releasing-hormone.

Chemical and enzymatic treatment of a high-molecular weight fraction from a frog brain extract resulted in formation of a “TRH-Like material” (TRH-i). Sequential treatment with trypsin and carboxypeptidases A and B, acetic acid and then chemical amidation generated a quantity of TRH-i equivalent to 25% of the endogenous TRH. TRH-i was similar to TRH (pGlu·His·ProNH₂) as assessed by molecular weight estimations, radioimmunoassay and susceptibility to serum inactivation. TRH and TRH-i also competed with [³H]-TRH for binding to TRH receptors, stimulated prolactin synthesis and uridine uptake, and “down-regulated” TRH receptors in pituitary cells. These results suggest the possibility that TRH may be processed from a macromolecular precursor.     

Horizontal gene transfer in microbial genome evolution

Horizontal gene transfer is the collective name for processes that permit the exchange of DNA among organisms of different species. Only recently has it been recognized as a significant contribution to inter-organismal gene exchange. Traditionally, it was thought that microorganisms evolved clonally, passing genes from mother to daughter cells with little or no exchange of DNA among diverse species. Studies of microbial genomes, however, have shown that genomes contain genes that are closely related to a number of different prokaryotes, sometimes to phylogenetically very distantly related ones. (Doolittle et al., 1990, J. Mol. Evol. 31, 383-388; Karlin et al., 1997, J. Bacteriol. 179, 3899-3913; Karlin et al., 1998, Annu. Rev. Genet. 32, 185-225; Lawrence and Ochman, 1998, Proc. Natl. Acad. Sci. USA 95, 9413-9417; Rivera et al., 1998, Proc. Natl. Acad. Sci. USA 95, 6239-6244; Campbell, 2000, Theor. Popul. Biol. 57 71-77; Doolittle, 2000, Sci. Am. 282, 90-95; Ochman and Jones, 2000, Embo. J. 19, 6637-6643; Boucher et al. 2001, Curr. Opin., Microbiol. 4, 285-289; Wang et al., 2001, Mol. Biol. Evol. 18, 792-800). Whereas prokaryotic and eukaryotic evolution was once reconstructed from a single 16S ribosomal RNA (rRNA) gene, the analysis of complete genomes is beginning to yield a different picture of microbial evolution, one that is wrought with the lateral movement of genes across vast phylogenetic distances. (Lane et al., 1988, Methods Enzymol. 167, 138-144; Lake and Rivera, 1996, Proc. Natl. Acad. Sci. USA 91, 2880-2881; Lake et al., 1999, Science 283, 2027-2028).     

Unusual carbon fixation gives rise to diverse polyketide extender units

Polyketides are structurally diverse and medically important natural products that have various biological activities. During biosynthesis, chain elongation uses activated dicarboxylic acid building blocks, and their availability therefore limits side chain variation in polyketides. Recently, the crotonyl-CoA carboxylase-reductase (CCR) class of enzymes was identified in primary metabolism and was found to be involved in extender-unit biosynthesis of polyketides. These enzymes are, in theory, capable of forming dicarboxylic acids that show any side chain from the respective unsaturated fatty acid precursor. To our knowledge, we here report the first crystal structure of a CCR, the hexylmalonyl-CoA synthase from Streptomyces sp. JS360, in complex with its substrate. Structural analysis and biochemical characterization of the enzyme, including active site mutations, are reported. Our analysis reveals how primary metabolic CCRs can evolve to produce various dicarboxylic acid building blocks, setting the stage to use CCRs for the production of unique extender units and, consequently, altered polyketides.     

An improved probability mapping approach to assess genome mosaicism

Background

Maximum likelihood and posterior probability mapping are useful visualization techniques that are used to ascertain the mosaic nature of prokaryotic genomes. However, posterior probabilities, especially when calculated for four-taxon cases, tend to overestimate the support for tree topologies. Furthermore, because of poor taxon sampling four-taxon analyses suffer from sensitivity to the long branch attraction artifact. Here we extend the probability mapping approach by improving taxon sampling of the analyzed datasets, and by using bootstrap support values, a more conservative tool to assess reliability.

Results

Quartets of orthologous proteins were complemented with homologs from selected reference genomes. The mapping of bootstrap support values from these extended datasets gives results similar to the original maximum likelihood and posterior probability mapping. The more conservative nature of the plotted support values allows to focus further analyses on those protein families that strongly disagree with the majority or plurality of genes present in the analyzed genomes.

Conclusion

Posterior probability is a non-conservative measure for support, and posterior probability mapping only provides a quick estimation of phylogenetic information content of four genomes. This approach can be utilized as a pre-screen to select genes that might have been horizontally transferred. Better taxon sampling combined with subtree analyses prevents the inconsistencies associated with four-taxon analyses, but retains the power of visual representation. Nevertheless, a case-by-case inspection of individual multi-taxon phylogenies remains necessary to differentiate unrecognized paralogy and shared phylogenetic reconstruction artifacts from horizontal gene transfer events.

     

Bacterial sugar utilization gives rise to distinct single‐cell behaviours

Inducible utilization pathways reflect widespread microbial strategies to uptake and consume sugars from the environment. Despite their broad importance and extensive characterization, little is known how these pathways naturally respond to their inducing sugar in individual cells. Here, we performed single‐cell analyses to probe the behaviour of representative pathways in the model bacterium Escherichia coli. We observed diverse single‐cell behaviours, including uniform responses (d ‐lactose, d ‐galactose, N‐acetylglucosamine, N‐acetylneuraminic acid), ‘all‐or‐none’ responses (d ‐xylose, l ‐rhamnose) and complex combinations thereof (l ‐arabinose, d ‐gluconate). Mathematical modelling and probing of genetically modified pathways revealed that the simple framework underlying these pathways – inducible transport and inducible catabolism – could give rise to most of these behaviours. Sugar catabolism was also an important feature, as disruption of catabolism eliminated tunable induction as well as enhanced memory of previous conditions. For instance, disruption of catabolism in pathways that respond to endogenously synthesized sugars led to full pathway induction even in the absence of exogenous sugar. Our findings demonstrate the remarkable flexibility of this simple biological framework, with direct implications for environmental adaptation and the engineering of synthetic utilization pathways as titratable expression systems and for metabolic engineering.     

Horizontal gene transfer, genome innovation and evolution

To what extent is the tree of life the best representation of the evolutionary history of microorganisms? Recent work has shown that, among sets of prokaryotic genomes in which most homologous genes show extremely low sequence divergence, gene content can vary enormously, implying that those genes that are variably present or absent are frequently horizontally transferred. Traditionally, successful horizontal gene transfer was assumed to provide a selective advantage to either the host or the gene itself, but could horizontally transferred genes be neutral or nearly neutral? We suggest that for many prokaryotes, the boundaries between species are fuzzy, and therefore the principles of population genetics must be broadened so that they can be applied to higher taxonomic categories.     

Phylogeny vs genome reshuffling: horizontal gene transfer

The evolutionary events in organisms can be tracked to the transfer of genetic material. The inheritance of genetic material among closely related organisms is a slow evolutionary process. On the other hand, the movement of genes among distantly related species can account for rapid evolution. The later process has been quite evident in the appearance of antibiotic resistance genes among human and animal pathogens. Phylogenetic trees based on such genes and those involved in metabolic activities reflect the incongruencies in comparison to the 16S rDNA gene, generally used for taxonomic relationships. Such discrepancies in gene inheritance have been termed as horizontal gene transfer (HGT) events. In the post-genomic era, the explosion of known sequences through large-scale sequencing projects has unraveled the weakness of traditional 16S rDNA gene tree based evolutionary model. Various methods to scrutinize HGT events include atypical composition, abnormal sequence similarity, anomalous phylogenetic distribution, unusual phyletic patterns, etc. Since HGT generates greater genetic diversity, it is likely to increase resource use and ecosystem resilience.     

Horizontal gene transfer accelerates genome innovation and evolution

Horizontal gene transfer (HGT) spreads genetic diversity by moving genes across species boundaries. By rapidly introducing newly evolved genes into existing genomes, HGT circumvents the slow step of ab initio gene creation and accelerates genome innovation. However, HGT can only affect organisms that readily exchange genes (exchange communities). In order to define exchange communities and understand the internal and external environmental factors that regulate HGT, we analyzed approximately 20,000 genes contained in eight free-living prokaryotic genomes. These analyses indicate that HGT occurs among organisms that share similar factors. The most significant are genome size, genome G/C composition, carbon utilization, and oxygen tolerance.     

Reducing the genome size of organelles favours gene transfer to the nucleus

Endosymbiotic organelles exhibit strong genetic erosion during their evolution as a result of the loss of unnecessary genes and of gene transfer to the nucleus. The reasons for this erosion are much debated. Unidirectionality of DNA exchange between cell compartments could favour biased gene transfer, but selection might also act to favour nuclear localization of genes, for example, because organelles accumulate more mutations than do nuclei. Selection for rapid replication might be a general cause of organelle genome reduction. This selection also accounts for the compactness of organelle genomes.     

Biochemical and genetic consequences of gene transfer from endosymbiont to host genome

Summary In the origin of the mitochondrion and plastid, gene transfer from the ancestral endosymbiont to the host was proposed to be a crucial event. For this genic integration to proceed, products of transferred genes had to return to and enter the endosymbionts. The limiting event was the crossing of the barrier presented by the two semipermeable membranes bounding the proto-organelle. In this paper it is suggested that spontaneous transport allowed transferred gene encoded proteins to enter the endosymbionts before receptors evolved. The effects of these events, including the degeneration of the endosymbiont genome, are discussed. Although the presumed gene transfer had profound effects on the metabolic relationships between host and endosymbionts it probably cannot account for all examples of organelle/cytoplasmic isozyme pairs or the absence of amino acid synthetic enzymes in animal cells.     

Finding pathogenicity islands and gene transfer events in genome data

MOTIVATION: There is a growing literature on wavelet theory and wavelet methods showing improvements on more classical techniques, especially in the contexts of smoothing and extraction of fundamental components of signals. G+C patterns occur at different lengths (scales) and, for this reason, G+C plots are usually difficult to interpret. Current methods for genome analysis choose a window size and compute a chi(2) statistics of the average value for each window with respect to the whole genome. RESULTS: Firstly, wavelets are used to smooth G+C profiles to locate characteristic patterns in genome sequences. The method we use is based on performing a chi(2) statistics on the wavelet coefficients of a profile; thus we do not need to choose a fixed window size, in that the smoothing occurs at a set of different scales. Secondly, a wavelet scalogram is used as a measure for sequence profile comparison; this tool is very general and can be applied to other sequence profiles commonly used in genome analysis. We show applications to the analysis of Deinococcus radiodurans chromosome I, of two strains of Helicobacter pylori (26695, J99) and two of Neisseria meningitidis (serogroup B strain MC58 and serogroup A strain Z2491). We report a list of loci that have different G+C content with respect to the nearby regions; the analysis of N. meningitidis serogroup B shows two new large regions with low G+C content that are putative pathogenicity islands. AVAILABILITY: Software and numerical results (profiles, scalograms, high and low frequency components) for all the genome sequences analyzed are available upon request from the authors.     

Alternative splicing of exon 3b gives rise to ODF2 and Cenexin

ODF2 was first identified as the major component of the sperm tail outer dense fibers. Additionally, ODF2 is a critical component of the mature centriole of the animal centrosome where it locates to the distal appendages. Moreover, generation of primary cilia strictly depends on ODF2. The mature centriole is characterized further by recruitment of Cenexin. Albeit highly similar in sequence the relationship between ODF2 and Cenexin has not been investigated. We demonstrate here that ODF2 and Cenexin are alternative splice products by identifying a novel exon 3b encoding Cenexin specific amino acids. Even though ODF2 is the main isoform in testicular tissue RT-PCR analyses revealed that isoforms are not restricted to specific tissues.     

Iron incorporation into Escherichia coli Dps gives rise to a ferritin-like microcrystalline core

Escherichia coli Dps belongs to a family of bacterial stress-induced proteins to protect DNA from oxidative damage. It shares with Listeria innocua ferritin several structural features, such as the quaternary assemblage and the presence of an unusual ferroxidase center. Indeed, it was recently recognized to be able to oxidize and incorporate iron. Since ferritins are endowed with the unique capacity to direct iron deposition toward formation of a microcrystalline core, the structure of iron deposited in the E. coli Dps cavity was studied. Polarized single crystal absorption microspectrophotometry of iron-loaded Dps shows that iron ions are oriented. The spectral properties in the high spin 3d(5) configuration point to a crystal form with tetrahedral symmetry where the tetrahedron center is occupied by iron ions and the vertices by oxygen. Crystals of iron-loaded Dps also show that, as in mammalian ferritins, iron does not remain bound to the site after oxidation has taken place. The kinetics of the iron reduction/release process induced by dithionite were measured in the crystal and in solution. The reaction appears to have two phases, with t(12) of a few seconds and several minutes at neutral pH values, as in canonical ferritins. This behavior is attributed to a similar composition of the iron core.     

Autolysis of proproteinase E in bovine procarboxypeptidase A ternary complex gives rise to subunit III

Extracts of bovine pancreatic tissue are shown by HPLC to contain two distinct ternary complexes of procarboxypeptidase A (subunit I), chymotrypsinogen C (subunit II) and either proproteinase E or subunit III. It is shown that proproteinase E in the complex generates subunit III by removal of 13 N-terminal residues when the former is allowed to autolyze in solution or when catalytic amounts of isolated active proteinase E are added to it. Autolysis of proproteinase E was accompanied by the loss of potential activity towards specific synthetic substrates and occurred at a higher rate in pancreatic juice than in pancreatic tissue extracts, even when both were processed in the presence of serine protease inhibitors. We conclude that subunit III (also called truncated protease E) is an autolytic product of proproteinase E and not an ab initio component of the native ternary complex.     

One type of gamma-turn, rather than the other gives rise to chain-reversal in proteins

Gamma-turns may be defined by a hydrogen bond between the carbonyl group of one amino acid residue and the amino group of the acid two residues ahead in the sequence. They occur as two types, inverse gamma-turns and classic gamma-turns (classic gamma-turns are usually called just gamma-turns but we prefer to add the adjective classic to distinguish them from the word gamma-turn, referring collectively to both). Of the two, classic gamma-turns are less common and are considered by all authors to be extreme rarities in proteins. However, we find that a number do occur in a sample of proteins of known three-dimensional structure. One occurs at the edge of the second hypervariable region of the light chain in some immunoglobulins. All classic gamma-turns except one are associated with a reversal in the main chain direction. In most cases, the turn lies at the loop end of a beta-hairpin. By contrast, inverse gamma-turns, although giving rise to a kink in the chain, rarely occur within beta-hairpins and are seldom situated at a position of reversal, by 180 degrees, in chain direction.