Rate matrices for analyzing large families of protein sequences.

We propose and study a new approach for the analysis of families of protein sequences. This method is related to the LogDet distances used in phylogenetic reconstructions; it can be viewed as an attempt to embed these distances into a multidimensional framework. The proposed method starts by associating a Markov matrix to each pairwise alignment deduced from a given multiple alignment. The central objects under consideration here are matrix-valued logarithms L of these Markov matrices, which exist under conditions that are compatible with fairly large divergence between the sequences. These logarithms allow us to compare data from a family of aligned proteins with simple models (in particular, continuous reversible Markov models) and to test the adequacy of such models. If one neglects fluctuations arising from the finite length of sequences, any continuous reversible Markov model with a single rate matrix Q over an arbitrary tree predicts that all the observed matrices L are multiples of Q. Our method exploits this fact, without relying on any tree estimation. We test this prediction on a family of proteins encoded by the mitochondrial genome of 26 multicellular animals, which include vertebrates, arthropods, echinoderms, molluscs, and nematodes. A principal component analysis of the observed matrices L shows that a single rate model can be used as a rough approximation to the data, but that systematic deviations from any such model are unmistakable and related to the evolutionary history of the species under consideration.     

A low-resolution model of crystalline methionyl-transfer RNA synthetase from Escherichia coli

When submitted to a controlled proteolysis by trypsin, native methionyl-tRNA synthetase from Escherichia coli (a dimer of molecular weight 172,000) yields a well-defined fragment of molecular weight 64,000 composed of one single polypeptide chain. This fragment retains full specificity towards methionine and tRNA^met, and has unimpaired activity in both the activation reaction and aminoacyl-tRNA formation. Crystals of this active fragment have been studied by X-ray crystallography and, using two isomorphous heavy-atom derivatives, a 4 Å electron density map has been calculated.The molecule appears as an elongated ellipsoid of overall dimensions 90 Å × 43 Å × 43 Å. It is clearly built of two parts separated by a large cleft. The volume of one of these “domains” is approximately twice that of the other; these results are consistent with our present knowledge of the chemistry of the protein.     

Response of vegetation and vertebrate fauna to 23 years of fire exclusion in a tropical Eucalyptus open forest,Northern Territory,Australia

Abstract This opportunistic study compares the vegetation, fuel loads and vertebrate fauna of part of a 120‐ha block of tropical open forest protected from fire for 23 years, and an adjacent block burnt annually over this period. Total fuel loads did not differ significantly between the unburnt and annually burnt sites, but their composition was markedly different, with far less grassy fuel, but far more litter fuel, in the unburnt block. There were major differences between treatments in the composition of trees and shrubs, manifest particularly in the number of stems. There was no overall difference in plant species richness between the two treatments, but richness of woody species was far higher in the unburnt treatment, and of annual and perennial grasses, and perennial herbs in the annually burnt treatment. Change in plant species composition from annually burnt to unburnt treatment was directional, in that there was a far higher representation of rainforest‐associated species (with the percentage of woody stems attributable to ‘rainforest’ species increasing from 24% of all species in the annually burnt treatment to 43% in the unburnt treatment, that of basal area from 9% to 30%, that of species richness from 8% to 17%, and that of cover from 12 to 47%). The vertebrate species composition varied significantly between treatments, but there was relatively little difference in species richness (other than for a slightly richer reptile fauna in the unburnt treatment). Again, there was a tendency for species that were more common in the unburnt treatment to be rainforest‐associated species. The results from this study suggest that there is a sizeable and distinct set of species that are associated with relatively long‐unburnt environments, and hence that are strongly disadvantaged under contemporary fire regimes. We suggest that such species need to be better accommodated by fire management through strategic reductions in the frequency of burning.     

Excreted steroids in primate feces over the menstrual cycle and pregnancy

Techniques were established for the extraction and measurement of 17 beta-estradiol (E2) and progestins (P4) from feces of Old World primates. Studies were conducted to show the sensitivity of these measures, means of preserving fecal samples in the field, effects of urinary contamination, and means to eliminate these effects. Our results show that excreted steroid measures can be used to distinguish between mid-follicular and luteal phases in the menstrual cycle, and to identify pregnancy by Day 20 of gestation; the steroid measures can also be used to identify ovulatory levels of E2 and to establish the length of the menstrual cycle. Urine was shown to contaminate the fecal sample and to confound the estimate of steroid levels in feces; prolonged storage (less than 6 h) was shown to change the steroid estimate. Both urinary contamination and storage-dependent changes were eliminated by the addition of ethanol to the sample. Preliminary results also suggest that effects of dietary fiber on steroid hormone levels are minimal when controlled quantitatively by adjusting for water content of the fecal sample. We conclude that these measurements of excreted steroids provide a valid, noninvasive measure of physiological state of the hypothalamic-pituitary-ovarian axis among free-ranging animals in the field.     

Evolution of genes,evolution of species: the case of aminoacyl-tRNA synthetases

All of the aminoacyl-tRNA synthetase (aaRS) sequences currently available in the data banks have been subjected to a systematic analysis aimed at finding gene duplications, genetic recombinations, and horizontal transfers. Evidence is provided for the occurrence (or probable occurrence) of such phenomena within this class of enzymes. In particular, it is suggested that the monomeric PheRS from the yeast mitochondrion is a chimera of the alpha and beta chains of the standard tetrameric protein. In addition, it is proposed that the dimeric and tetrameric forms of GlyRS are the result of a double and independent acquisition of the same specificity within two different subclasses of aaRS. The phylogenetic reconstructions of the evolutionary histories of the genes encoding aaRS are shown to be extremely diverse. While large segments of the population are consistent with the broad grouping into the three Woesian domains, some phylogenetic reconstructions do not place the Archae and the Eucarya as sister groups but, rather, show a gram-negative bacteria/eukaryote clustering. In addition, many individual genes pose difficulties that preclude any simple evolutionary scheme. Thus, aaRS's are clearly a paradigm of F. Jacob's "odd jobs of evolution" but, on the whole, do not call into question the evolutionary scenario originally proposed by Woese and subsequently refined by others.     

Crystallographic study at 2.5 A resolution of the interaction of methionyl-tRNA synthetase from Escherichia coli with ATP

The crystal structure of the tryptic fragment of the methionyl-tRNA synthetase from Escherichia coli, complexed with ATP, has been refined to a crystallographic R-factor of 0.220, at 2.5 A resolution (for 4433 protein atoms). In the last stages of the refinement, the simulated annealing refinement method was fully applied, contributing to a drastic improvement of the model and the identification of the missing atoms. In the final model, the root-mean-square deviation from ideality for bond distances is 0.021 A and for angle distances is 0.054 A. The position of the zinc ion has been confirmed and is located near the active site. The tryptic fragment is composed of two globular domains. The first domain, from the N terminus to Thr360, contains a nucleotide-binding fold into which two long polypeptides of 101 and 70 residues are inserted. The nucleotide-binding fold is strengthened by the presence of the zinc ion in the vicinity of the active site. The second domain, up to Pro526, is mainly alpha-helical. The C-terminal polypeptide, Phe527 to Lys551, folds back towards the first domain, making a link between the two domains. The heptapeptide 528-534 partly shapes a deep cavity that plunges into the central core of the nucleotide-binding fold, where the ATP molecule is located. The adenine ring, deeply buried in the bottom of the cleft, is blocked between the first helix HA, and the strands A and D of the beta-sheet and makes no polar interaction with the enzyme. The 2' and 3' hydroxyl groups of the ribose, whose conformation is C2' endo, interact with the main-chain carbonyl oxygen atoms of Ile231 and Glu241, respectively. The side-chain nitrogen atom of Lys142 is at hydrogen-bonding distance from the ring oxygen O-4' of the ribose. One of the alpha-phosphate oxygen atoms and one of the gamma-phosphate oxygen atoms interact with the imidazole ring of His21, which is well conserved in many of the known synthetases; this indicates a possible crucial role for this residue in binding ATP. The beta-phosphate group is linked to the main-chain carbonyl oxygen atom of Tyr15 through an intermediate water molecule. The gamma-phosphate group interacts with the carbonyl oxygen atom and the side-chain of Asn17.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H. anomala proteinase

The protomeric chain of Hansenula anomala flavocytochrome b2 was previously shown to be built as the covalent association of two functional domains: an L-lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H. anomala proteinase(s) preparation and the purification of the resulting L-lactate dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer (Mr = 4 x 39000) containing FMN as expected and not heme. It has high L-lactate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2) and the same Km for L(+)-lactate as flavocytochrome b2, but it has no L-lactate:cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochrome b2. The subcellular origin of the H. anomala proteinase in the preparation has not yet been elucidated.     

Non Mycobacterial Virulence Genes in the Genome of the Emerging Pathogen Mycobacterium abscessus

Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycobacterium marinum. The chromosome encodes many virulence proteins and virulence protein families absent or present in only small numbers in the model RGM species Mycobacterium smegmatis. Many of these proteins are encoded by genes belonging to a “mycobacterial” gene pool (e.g. PE and PPE proteins, MCE and YrbE proteins, lipoprotein LpqH precursors). However, many others (e.g. phospholipase C, MgtC, MsrA, ABC Fe(3+) transporter) appear to have been horizontally acquired from distantly related environmental bacteria with a high G+C content, mostly actinobacteria (e.g. Rhodococcus sp., Streptomyces sp.) and pseudomonads. We also identified several metabolic regions acquired from actinobacteria and pseudomonads (relating to phenazine biosynthesis, homogentisate catabolism, phenylacetic acid degradation, DNA degradation) not present in the M. smegmatis genome. Many of the “non mycobacterial” factors detected in M. abscessus are also present in two of the pathogens most frequently isolated from CF patients, Pseudomonas aeruginosa and Burkholderia cepacia. This study elucidates the genetic basis of the unique pathogenicity of M. abscessus among RGM, and raises the question of similar mechanisms of pathogenicity shared by unrelated organisms in CF patients.     

Beef liver L-glutamate dehydrogenase mechanism: Presteady state study of the catalytic reduction of 2.oxoglutarate by NADPH

The mechanism of the reduction of 2.oxoglutarate by NADPH and addition of NH₄⁺ catalyzed by L-glutamate dehydrogenase was investigated with the stopped flow technique, following dihydronicotinamide absorbance and fluorescence changes. We make conspicuous, as in the reverse reaction, the existence of prestationnary events, the characteristics of which strongly depend on the ligand addition order. If the E-NADPH-2.oxoglutarate complex is preformed, saturated with regard to each ligand, there is a burst associated with the oxidation of 0.7 mole of NADPH per mole of protomer, a first order process with a velocity constant of 300 ± 50 sec^?1. If this complex is not preformed, the burst has reduced amplitude and velocity constants, differences that suggest an isomerization step following the binding processes. Whichever the incubation order, the later evolution follows an exponential decrease towards equilibrium with a velocity constant depending on the ligand concentration up to an extrapolated value of 27 sec^?1, a step corresponding to the dissociation of the reaction products.